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25-8010-38UM Rev B 2006.indd - GE Healthcare Life Sciences

1. Control no IL 6 30 ng ml IL 6 5 4 11 AG490 inhibition curve Fig 5 13 shows an inhibition curve for the antagonist AG490 The data were collected 25 minutes after the addition of control no IL 6 or 30 ng ml IL 6 to cells which had been pre incubated with varying concentrations of AG490 for 4 hours An ICsg of 20 uM was obtained Data were acquired from a single experiment with 5 uM Hoechst nuclear stain as opposed to the recommended 1 UM Hoechst Control no IL 6 ja a 30 ng mlig Fig 5 13 AG490 inhibition curve in the presence of Control no IL 6 or amp j 30 ng ml IL 6 Error SD n 4 F z replicates per data point IC5g 20 uM EEk 52 Z E 14 0 T T T T 1 8 7 6 5 4 3 log AG490 M 25 8010 38UM Chapter 5 Rev B 2006 23 5 4 12 Results obtained on the IN Cell Analyzer 1000 Fig 5 14 and Fig 5 15 show typical images and data for the EGFP STAT3 translocation assay After 25 minutes incubation in the absence or presence of 30 ng ml IL 6 cells were fixed with 2 formaldehyde in PBS prior to imaging on the IN Cell Analyzer 1000 Fig 5 14 Images obtained using IN Cell Analyzer 1000 The BHK derived EGFP STAT3 expressing cells 25 minutes after stimulation with A control buffer only or B 30 ng ml IL 6 Fig 5 15 Results obtained using the IN Cell Analyzer 1000 and associated 1 4 Nuclear Trafficking Analysis Module x lt oO e O ai Cy co
2. y Decant Wash Decant Add Assay medium with nuclear stain y Incubate 30 minutes 37 C 5 CO2 y Add control and test compounds y Incubate 25 minutes 37 C 5 CO2 AA Image plate on IN Cell Analyzer 3000 STOP 25 8010 38UM Chapter 5 Rev B 2006 16 placed into the IN Cell Analyzer 3000 The Nuclear Trafficking Analysis Module is used to analyze images from each well 5 2 4 Agonist assay protocol 96 well format NOTE whenever possible keep the microplate at 37 C 5 CO and 95 humidity 1 The day before starting the assay seed 0 8 x 104 cells per well in 200 ul of Growth medium Incubate for 24 hours at 37 C 5 COs If one of the wells on the cell plate is used for flat field correction it should not contain cells 2 On the day of the assay prepare the test compounds solvent controls if used and reference agonist control IL 6 These samples are typically prepared at four fold of the final concentration in Assay medium For the reference agonist IL 6 a final concentration of 30 ng ml is suitable However we recommend that users perform their own dose response curve to establish optimal agonist concentrations The concentration range of IL 6 recommended for a dose response assay is 0 3 ng ml to 1 ug ml 3 Decant the Growth medium from the cell plate removing all excess liquid and add 100 ul Assay medium to wash the cells Decant the wash 4 Ad
3. 25 8010 38UM Chapter 5 Rev B 2006 22 5 4 9 Effect of using DRAQ5 nuclear stain GFP expressing cells which have been stained using Hoechst nuclear marker must be imaged sequentially due to overlap of spectral profiles for the two probes For speed of imaging the red nuclear marker DRAQ5 can be used instead of Hoechst Since there is no spectral overlap between DRAQ5 and GFP images can be acquired simultaneously Fig 5 11 shows an IL 6 dose response curve where the nuclear marker has been changed to 0 3 uM DRAQS ad Fig 5 11 IL 6 dose response curve using 0 3 UM DRAQS as the nuclear marker Error SD n 8 replicates per N i data point ECsg 3 9 ng ml 5S 0 T T T T 1 2 1 0 1 2 3 log IL 6 ng ml 5 4 10 Fixed Assay In order to avoid the need for strict time controlled dispensing it is possible to complete the assay using live cells as described in the agonist assay protocol but fix the cells prior to analysis For full details see section 5 2 5 Fig 5 12 shows results obtained from fixed preparations after 25 minutes stimulation with control no IL 6 or 30 ng ml IL 6 Cells were fixed with 2 formaldehyde in PBS Fig 5 12 IL 6 induced EGFP STAT3 translocation Cells treated with either Control no IL 6 or 30 ng ml IL 6 for x Bs 25 minutes prior to fixation and 5 fs imaging on the IN Cell Analyzer 3000 S5 Error SD n 48 replicates per data 22 3 E ad point
4. 4824 5304 c 5557 5758 6944 7114 c 7383 7426 7544 7950 8062 c 8075 c BssHII 1 6010 BstBl 1 6295 BstNl 27 244 437 1153 1278 1390 1465 1519 1897 2035 2236 2359 2442 3273 3354 3458 3583 3662 3850 3928 3950 5133 5188 5205 6000 8318 8331 8452 BstUl 21 214 1436 1754 1851 4507 4531 4551 4927 5014 5679 5980 6012 6413 6493 6596 6598 6698 7030 7523 7853 8434 Bstx 3 3272 3849 6334 BstY 17 1607 1834 2322 2412 2992 3752 5016 5784 6030 6407 6942 6959 7727 7739 7825 7836 8489 Bsu36l 1 3091 Cfol 34 1092 1397 1438 1754 2481 3955 4147 4485 4509 4522 4531 4553 4579 4587 5024 5607 5615 5679 5716 5982 6012 6014 6242 6495 6598 6698 7030 7367 7460 7853 7962 8136 8236 8303 Cfr10l 9 1095 1258 2756 2855 2875 4630 5932 6113 7504 Clal 2 4425 6394 Csp45l 1 6295 Csp6l 18 98 372 452 485 536 701 1063 1536 1818 1893 2541 3291 3576 3603 5070 5918 6431 7107 Dadel 21 1711 1729 1831 2921 2986 3091 3152 3376 3412 3653 3676 4089 5077 5379 6276 6427 6662 7088 7628 7794 8203 Dpnl 40 664 749 1609 1757 1795 1836 2324 2414 2636 2994 3169 3244 3359 3484 3754 3829 4424 4428 4464 5018 5786 5864 5945 5954 6032 6409 6908 6944 6961 7219 7265 7283 7624 7729 7741 7819 7827 7838 7913 8491 Dpnil 40 662 747 1607 1755 1793 1834 2322 2412 2634 2992 3167 3242 3357 3482 3752 3827 4422 4426 4462 5016 5784 5862 5943 5952 6030 6407 6906 6942 6959 7217 7263 7281 7622 7727 7739 7817 7825 7836 7911 8489 Dral 4 4380 7011 7703 7722 Drall 4 2739 3372 3454 6609 Dralll 1 4738 Drdl 6 818 47
5. Fixed cell assay format In order to avoid the need for strict time controlled dispensing of test and control compounds it is possible to complete the assay using live cells as described in the agonist assay protocol but fix the cells prior to imaging 1 Perform the assay as described 2 After incubating with agonist for the optimal time decant the Assay medium and wash the cells in each well with 200 ul PBS Decant the wash 3 Add 100 ul 2 formaldehyde in PBS and incubate for 30 minutes at room temperature 4 Decant the fixative and wash the cells in each well with 200 ul PBS Decant the wash 5 Store the plate at 4 C with 100 ul fresh PBS in each well 6 Image and perform analysis as required If the cells are to be fixed the assay can be performed with the Nuclear stain in the Assay medium for the duration of the assay Nuclear staining at the same 25 8010 38UM Chapter 5 Rev B 2006 17 concentrations as normal can be performed after fixation If no stain is used during the assay the first PBS wash step can be omitted from the protocol 5 3 Results 5 3 1 Calculating the 2 factor Assay performance can be assessed by calculating the Z factor a dimensionless value defined by Zhang et al 32 Using the IN Cell Analyzer 3000 a Z factor of gt 0 3 should be obtained with the assay under standard conditions if the experiment is performed as described in this manual 30c 30c e pe w
6. 1197 c 1203 c 1297 1434 c 1446 c 1497 c 1617 c 2398 2559 c 2579 c 2605 c 2700 c 2763 c 2872 2983 c 3073 3086 c 3147 c 3177 c 3211 3363 c 3400 c 3402 3668 3697 c 3885 c 3888 c 4090 c 4098 4146 4161 4364 c 4404 4444 c 4708 5048 c 5056 5072 c 5350 c 5356 c 5380 5386 5393 c 5396 c 5408 c 5528 c 5664 c 6021 c 6214 6563 c 6622 7216 c 7422 c 7569 7650 8050 8300 c 8374 Mrol 3 1825 2306 2850 Mscl 3 11 65 5695 Msel 34 161 784 830 849 917 1052 1067 2696 2800 2939 2945 3005 3301 4114 4318 4379 4525 4796 4894 4911 4922 4934 4945 5468 6457 6638 7010 7375 7414 7649 7702 7716 7721 7773 Msll 17 519 1108 1138 1288 1465 1594 1852 2017 2842 3028 3721 6050 6332 6371 6818 7177 7336 MspA1l 10 2460 3426 3740 3786 5095 5719 6487 6953 7894 8139 Mspl 36 1096 1136 1199 1259 1790 1826 2160 2172 2307 2757 2851 2856 2876 2997 4171 4430 4631 5518 5595 5617 5645 5776 5866 5933 6114 6517 6551 7052 7294 7404 7471 7505 7909 8099 8125 8272 Munl 2 3344 4328 Mval 27 244 437 1153 1278 1390 1465 1519 1897 2035 2236 2359 2442 3273 3354 3458 3583 3662 3850 3928 3950 5133 5188 5205 6000 8318 8331 8452 Mvnl 21 214 1436 1754 1851 4507 4531 4551 4927 5014 5679 5980 6012 6413 6493 6596 6598 6698 7030 7523 7853 8434 Mwol 53 137 244 366 398 437 530 554 803 1054 1199 1259 1272 1316 1325 1878 1911 1990 2215 25 8010 38UM Chapter 11 2218 2280 2289 2451 2478 2520 2612 2757 2838 4464 4494 4526 4528 4570 4597 4627 Rev B 2006 35 Enzyme of c
7. 9 730 1717 3652 5726 5916 6424 6921 7006 8167 Aspl 1 5 31 Asull al 6295 Aval 2 1838 4170 Avall 9 1764 2484 2739 2853 3372 3454 6129 7225 7447 Avill 4 4484 5023 5715 7366 25 8010 38UM Chapter 11 Rev B 2006 31 Enzyme of cuts Positions c indicates the complementary strand Avril 1 5419 BamHI 2 2992 6407 Banl 11 619 977 1143 1892 2174 3664 4694 5069 5612 5647 7636 Banll 5 730 4664 5978 Bbsl 2 962 3976 Bbvl 45 821 c 1253 c 1359 1643 1650 1676 c 1679 c 1890 1914 1928 c 2221 2266 2286 2301 2466 2469 2727 2763 2841 2992 3071 3146 c 3435 3704 3792 3795 3942 c 4016 4290 c 4497 4565 5036 5560 c 5686 5728 5744 c 5837 c 6249 6544 c 7155 c 7546 7849 c 8055 c 8058 c 8148 Bcgl 2 1232 7051c Bcll 1 3167 Bfal 15 154 753 1058 1087 2326 2632 2641 3214 4231 4582 5420 5474 7396 7731 7984 Bfrl 4 829 848 1051 5467 Bgll 8 137 244 366 437 2218 4494 5372 7471 Bglll 3 1834 2412 8489 Binl 1 5419 Bmyl 17 730 1148 1277 1526 1717 3019 3652 4664 5559 5652 5726 5916 5978 6424 6921 7006 8167 Bpml 9 1552 1792 1918 2305 2341 c 2725 2788 3644 c 7520 BpuAl 2 962 3976 BsaAl 4 494 2544 4735 5917 BsaBl 2 4421 6406 BsaHl 11 276 329 412 598 2879 3277 3707 5613 6315 6667 7049 Bsal 4 916 c 3172 c 3211c 7523 BsaJ 30 514 1106 1136 1276 1439 1463 1518 1960 2034 2258 3182 3195 3272 3353 3368 3761 3887 3948 4136 4170 5030 5131 5203 5326 5361 5370 5419 5776 6045 8317 BsaWl 8 1095 1825 2306 2850 5644 7293 8124 8271 Bse
8. an enhancer element in the promoter of acute phase genes 7 8 In fact given the correct physiological context STAT3 is activated not only by IL 6 but by the entire family of IL 6 type cytokines including IL 11 leukemia inhibitory factor ciliary neurotrophic factor oncostatin M cardiotrophin 1 and cardiotrophin like cytokine 9 Non cytokine ligands such as epidermal growth factor hepatocyte growth factor platelet derived growth factor granulocyte colony stimulating factor and leptin have all been shown to activate STAT3 3 10 Upon stimulation of cell surface receptors STAT3 proteins are recruited to activated receptors through an interaction between the STAT3 src homology 2 SH2 domain and phosphotyrosine docking sites on the receptors intracellular domains Subsequently STAT3 is phosphorylated at a single site close to the carboxy terminus Tyr 5 either directly by the receptor kinase or indirectly by a receptor associated Janus kinase Jak Following phosphorylation STAT3 forms a dimer in which the SH2 domain of one STAT3 molecule binds to the phosphorylated Tyr 05 of the other and vice versa The STAT3 dimer then translocates through the cytoplasm to the nucleus 4 The processes by which this occurs are poorly understood although receptor mediated endocytosis may be involved 11 Within the nucleus STAT3 dimers recognize and bind an 8 10 base pair inverted repeat DNA element with a consensus sequence 5 TT N4 6 AA 3 com
9. in a suitably equipped laboratory environment Users within the jurisdiction of the European Union are bound by the provisions of European Directive 98 81 EC which amends Directive 90 219 EEC on Contained Use of Genetically Modified Micro Organisms These requirements are translated into local law which MUST be followed In the case of the UK this is the GMO Contained Use Regulations 2000 Information to assist users in producing their own risk assessments is provided in sections 3 3 1 and 3 3 2 of The Genetically Modified Organisms contained use Regulations 2000 http www legislation hmso gov uk si si2000 20002831 htm Risk assessments made under The Genetically Modified Organisms Contained Use Regulations 2000 for our preparation and transport of these cells indicate that containment 1 is necessary to control risk This risk is classified as GM Class 1 lowest category in the United Kingdom For handling precautions within the United States consult the National Institute of Health s Guidelines for Research Involving Recombinant DNA Molecules Instructions relating to the handling use storage and disposal of genetically modified materials 1 These components are shipped in liquid nitrogen vapor To avoid the risk of burns extreme care should be taken when removing the samples from the vapor and transferring to a liquid nitrogen storage unit When removing the cells from liquid nitrogen storage and thawing there is the
10. in section 5 1 5 3 Pellet the cells at approximately 300 g for 5 minutes Aspirate the medium from the cells 4 Gently resuspend the cells until no clumps remain in Freeze medium at a concentration of 1 x 108 cells in 1 ml and transfer into cryo vials Each vial should contain 1 x 106 cells in 1 ml of Freeze medium 5 Transfer the vials to a cryo freezing device and freeze at 80 C for 16 24 hours 6 Transfer the vials to the vapor phase in a liquid nitrogen storage device 5 1 7 Growth characteristics Under standard growth conditions the cells should maintain an average size of 18 6 um as measured using a CASY1 Cell Counter and Analyzer System Model TT The doubling time of the cell line in exponential growth phase has been determined to be approximately 13 4 hours under standard conditions Fig 5 1 The growth rate may decrease after having undergone more then 10 passages 25 8010 38UM Chapter 5 Rev B 2006 15 12 Fig 5 1 Growth curve of the 104 BHK derived EGFP STAT3 expressing cell line Only points on the linear portion are shown Doubling time Ba 13 4 hours In cell number gt gt 0 25 50 75 100 125 Time hours 5 2 Assay set up 5 2 1 Live cell EGFP STAT3 assay using the IN Cell Analyzer 3000 This manual provides a suggested protocol to use the EGFP STAT3 assay for agonist screening on the IN Cell Analyzer 3000 5 2 2 Microplate set up for 96 well format assays The EGFP STAT3 assay is
11. om nie SS oz 1 0 0 8 Control no IL 6 30 ng ml IL 6 25 8010 38UM Chapter 5 Rev B 2006 24 6 Vector use details The plasmid vector pCORON1000 EGFP STAT3 Fig 3 1 can be used to transiently or stably express EGFP STAT3 fusion protein in the cell line of choice 6 1 General guidelines for vector use pCORON1000 EGFP STAT3 has been used successfully to express EGFP STAT3 fusion protein both transiently and stably in the BHK derived cell line Expression levels translocation responses and other assay parameters may vary depending on the cell type and the transfection procedure 6 2 Transient transfection with pbCORON1000 EGFP STAT3 Transient transfection protocols must be optimized for the cell type of choice Choice of transfection reagent and cell type will affect efficiency of transfection FuGENE 6 Transfection Reagent Roche produced successful results when transfecting pCORON1000 EGFP STAT3 into BHK cells For more information refer to manufacturer s guidelines for the desired transfection reagent 6 3 Stable cell line generation with PCORON1000 EGFP STAT3 The process of establishing stable cell lines involves a large number of variables many of which are cell line dependent Standard methods and guidelines for the generation of stable cell lines are widely available in the public domain 33 pCORON1000 EGFP STAT3 has been used to generate stably transfected cell populations The magnitude o
12. possibility of an increase in pressure within the vial due to residual liquid nitrogen being present Appropriate care should be taken when opening the vial 2 Genetically modified cells supplied in this package are for use in a suitably equipped laboratory environment and should be used only by responsible persons in authorized areas Care should be taken to prevent ingestion or contact with skin or clothing Protective clothing such as laboratory overalls safety glasses and gloves should be worn whenever genetically modified materials are handled 3 Avoid actions that could lead to the ingestion of these materials and NO smoking drinking or eating should be allowed in areas where genetically modified materials are used 4 Any spills of genetically modified material should be cleaned immediately with a suitable disinfectant 25 8010 38UM Chapter 4 Rev B 2006 11 5 Hands should be washed after using genetically modified materials 6 Care should be taken to ensure that the cells are NOT warmed if they are NOT being used immediately To maintain viability DO NOT centrifuge the cells upon thawing 7 Most countries have legislation governing the handling use storage disposal and transportation of genetically modified materials The instructions set out above complement Local Regulations or Codes of Practice Users of these products MUST make themselves aware of and observe relevant Local Regulations or Codes of Practice For
13. the conditions described in the legend to Fig 5 3 25 8010 38UM Chapter 5 Rev B 2006 18 5 4 Assay characterization 5 4 1 Translocation index All of the characterizations for the EGFP STAT3 assay were performed on the IN Cell Analyzer 3000 using the Nuclear Trafficking Analysis Module The translocation index for this assay is taken to be the Nuc Cyt ratio reported for the cell population imaged in each well The Nuc Cyt ratio is the population averaged ratio of sampled nuclear and cytoplasmic intensities measured in the signal channel This index is used in all the following data 5 4 2 Summary of quantitative assay parameters Summaries of typical assay data using IL 6 as the agonist are shown in Tables 5 1 and 5 2 In particular Table 5 1 shows the results calculated from a single assay plate indicating the degree of well to well variation Table 5 2 shows a summary of the results obtained from 19 assays performed by different operators on different occasions giving an indication of inter assay variation Parameter Assay Data Assays Replicates Signal to Noise 42 46 1 48 Z factor 0 65 1 48 Magnitude of Response 1 25 1 48 CV Stimulated 4 77 1 48 Unstimulated 2 57 1 48 Parameter Assay Data SD Assays Replicates Signal to Noise 33 93 7 22 17 48 Z factor 0 51 0 10 17 48 Magnitude of Response 1 10 0 14 17 48 CV Stimulated 6 32 1 20 17 48 Unstimulated 2 79 0 36 17 48 25 8010 38UM Chap
14. 0 1c 2314 2374 2834 c 3268 c 3565 c 3847 3920 c 5270 c 5937 5962 6507 c 7150 7437 7618 Fspl 4 4484 5023 5715 7366 Haell 6 1093 3956 4580 4588 5616 8237 Haelll 46 11 65 238 431 1181 1281 1570 1682 1858 1957 1993 2170 2221 2234 2239 2615 2760 2859 3119 3200 3367 3586 3640 3918 4178 4454 4743 4885 5035 5360 5366 5375 5418 5521 5695 6086 6113 6338 6611 7198 7465 7545 8003 8437 8455 8466 Hgal 10 688 2868 c 3266 c 3696 c 4513 6323 6499 7057 7787 c 8365 c HgiAl 9 730 1717 3652 5726 5916 6424 6921 7006 8167 Hhal 34 1092 1397 1438 1754 2481 3955 4147 4485 4509 4522 4531 4553 4579 4587 5024 5607 5615 5679 5716 5982 6012 6014 6242 6495 6598 6698 7030 7367 7460 7853 7962 8136 8236 8303 HinP1l 34 1090 1395 1436 1752 2479 3953 4145 4483 4507 4520 4529 4551 4577 4585 5022 5605 5613 5677 5714 5980 6010 6012 6240 6493 6596 6696 7028 7365 7458 7851 7960 8134 8234 8301 Hincll 7 678 2689 3302 4115 4133 4167 4319 Hindll 7 678 2689 3302 4115 4133 4167 4319 Hindlll 2 757 5435 Hinfl 30 564 842 958 1074 1829 1866 1971 2002 2074 2108 2188 2352 2394 2668 2777 2965 3046 3680 4045 4108 4163 4783 4805 5441 6098 6232 6284 6391 7590 8107 Hpal 5 3302 4115 4319 Hpall 36 1096 1136 1199 1259 1790 1826 2160 2172 2307 2757 2851 2856 2876 2997 4171 4430 25 8010 38UM Chapter 11 4631 5518 5595 5617 5645 5776 5866 5933 6114 6517 6551 7052 7294 7404 7471 7505 7909 8099 8125 8272 Rev B 2006 34 Enzyme of cuts Positions c indicates the complementa
15. 10 v v FBS 1 v v Penicillin Streptomycin and 0 5 mg ml Geneticin e Freeze medium DMEM with Glutamax 1 supplemented with 10 v v FBS 1 v v Penicillin Streptomycin and 10 v v DMSO e Assay medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 mM HEPES 0 2 w v BSA and 1 0 uM Hoechst Nuclear stain 25 8010 38UM Chapter 5 Rev B 2006 13 e 10 ug ml IL 6 IL 6 reconstituted as recommended by the supplier Add 1 ml of sterile PBS containing 0 1 BSA to 10 ug IL 6 The stock solution of 10 ug ml IL 6 can be aliquoted and stored between 4 C and 70 C until required As recommended by the supplier stock solutions are stable for 1 month at 4 C or for 3 months at 70 C On the day of the assay the stock solution of IL 6 is further diluted in Assay medium for use in the assay see section 5 2 4 for further details e Flat field FF solution components e Cy5 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS e Oregon Green 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS e Alexa Fluor 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS As explained in the IN Cell Analyzer 3000 user manual prepare the FF solution to give adequate fluorescent signal in each channel used where the fluorescent counts should be less than 3300 at maximum For a Hoechst 33342 nuclear stained assay prepare an initial FF solution containing 1 0 ul 10 uM Oregon Green and 20 ul 100 u
16. 2542 3292 3577 3604 5071 5919 6432 7108 Rsrlk 2 2853 6129 Sacl al 730 Sall 1 4165 Sap 2 5957 c 6167 c Sau3Al 40 662 747 1607 1755 1793 1834 2322 2412 2634 2992 3167 3242 3357 3482 3752 3827 4422 4426 4462 5016 5784 5862 5943 5952 6030 6407 6906 6942 6959 7217 7263 7281 7622 7727 7739 7817 7825 7836 7911 8489 Sau96l 28 237 430 1280 1681 1764 1857 1956 2169 2220 2232 2484 2614 2739 2853 2858 3117 25 8010 38UM Chapter 11 3372 3454 3585 3638 4453 4741 6129 6609 7225 7447 7464 7543 Rev B 2006 36 Enzyme of cuts Scal 2 ScrFl 42 SexAl 2 SfaNl 35 Sfcl 13 Sfil 1 Sful 1 Smal 1 SnaBl 1 Snol 3 Spel 1 Sphl 4 SspBl 2 Ssp 4 Stul 2 Styl 9 Taql 25 Tfil 14 Thal 21 Tru9 34 Tsp5091 24 Tth1111 1 Van9 11 2 Xbal 1 xcm 2 Xhol 1 Xhol 17 Xma 1 xmalll 5 Xmn 1 25 8010 38UM Chapter 11 Positions c indicates the complementary strand 1064 7108 244 437 1137 1153 1278 1390 1465 1519 1791 1897 2035 2160 2172 2236 2359 2442 2997 3273 3354 3458 3583 3662 3850 3928 3950 4171 4172 4431 5133 5188 5205 5617 5777 6000 6517 6552 7053 7404 8100 8318 8331 8452 3456 5186 511 c 1206 c 1484 1499 1598 2270 c 2441 c 2459 c 2480 c 2510 2753 c 2856 3123 c 3251 c 3425 3825 c 3942 4043 c 4262 c 4954 c 4994 5176 5248 5571 c 5826 c 5912 5976 6042 c 6251 6435 c 6529 6888 c 7137 7328 c 8380 c 835 1080 1873 1917 2200 2207 3068 4002 4512 5662 7343 8021 8212 5372 6295 4172 494 6420 6917 8163 153 28
17. 332 415 601 6670 Acc 5l 2 1892 5069 Accl 1 4166 Acclll 3 1825 2306 2850 Acil 90 129 212 240 252 266 399 433 524 c 557 c 669 690 c 767 c 1326 1367 1434 1473 1611 1724 1784 1787 1944 c 1990 c 2061 2218 c 2493 c 2518 c 2589 c 2617 2826 c 3630 c 3636 c 3740 c 4035 4175 c 4179 4456 4517 c 4531 c 4534 c 4562 4589 4967 c 4993 c 5006 5014 c 5082 c 5267 5279 5288 5300 5310 5321 5367 5522 5585 5679 c 5743 c 5844 c 5847 c 6087 6127 c 6132 6182 c 6198 6224 6280 c 6339 6411 6449 6475 6485 6524 6698 c 6745 6844 c 6953 c 7030 c 7074 7195 c 7241 7432 c 7523 c 7885 7894 c 8029 8139 c 8260 c 8279 c 8406 c 843 4 c Acs 8 1843 2914 3001 3073 3496 4264 4918 4929 Acyl 11 276 329 412 598 2879 3277 3707 5613 6315 6667 7049 Aflll 4 829 848 1051 5467 Afllll 5 1849 3296 3794 Agel 1 1095 Alul 46 728 759 834 1048 1128 1161 1233 1266 1482 1530 1641 1815 1872 1881 1905 1914 1928 1941 2460 2514 2571 2787 2937 2983 3159 3380 3426 3786 3943 4303 4648 4905 5095 5383 5437 5719 6177 6538 6557 7236 7299 7399 7920 8177 8223 8313 Alw44l 3 6420 6917 8163 Alwl 23 1602 c 1801 2330 2987 c 3000 3365 3747 c 4421 c 4430 5024 5792 5857 c 6038 6402 c 6415 6950 6954 c 7271 7734 c 7735 7831 c 7833 7919 AlwNI 7 1953 2215 2283 2460 3786 3955 8068 Aosl 4 4484 5023 5715 7366 ApaLl 3 6420 6917 8163 Apol 8 1843 2914 3001 3073 3496 4264 4918 4929 Asel 2 161 7414 Asnl 2 161 7414 Asp700 1 6989 Asp718 2 1892 5069 AspEl 1 7589 AspHI
18. 41 5167 5239 6018 97 1817 6 53 4943 6784 3200 5418 514 1106 1960 3195 3887 5030 5326 5419 6045 824 945 1157 1451 1478 1493 1622 1839 2077 3419 4166 4184 4425 4700 5462 5726 5882 5906 5942 6104 6295 6394 6935 8379 8484 1866 2002 2108 2188 2352 2668 2777 3046 3680 4045 5441 6098 6232 6391 214 1436 1754 1851 4507 4531 4551 4927 5014 5679 5980 6012 6413 6493 6596 6598 6698 7030 7523 7853 8434 161 784 830 849 917 1052 1067 2696 2800 2939 2945 3005 3301 4114 4318 4379 4525 4796 4894 4911 4922 4934 4945 5468 6457 6638 7010 7375 7414 7649 7702 7716 7721 7773 172 786 1040 1843 2693 2914 2928 2942 3001 3073 3344 3465 3496 4264 4328 4918 4929 4955 5173 5245 5337 7156 7411 7717 5731 3287 3762 2325 2256 3350 1838 1607 1834 2322 2412 2992 3752 5016 5784 6030 6407 6942 6959 7727 7739 7825 7836 8489 4170 2758 4176 5519 6989 Rev B 2006 37 GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan GE Healthcare regional office contact numbers Asia Pacific Tel 85 65 6 275 1830 Fax 852 2811 5251 Australasia
19. 82 5456 5640 6506 8375 Dsal 7 514 1106 2258 3761 5030 5326 6045 DsaV 42 242 435 1135 1151 1276 1388 1463 1517 1789 1895 2033 2158 2170 2234 2357 2440 25 8010 38UM Chapter 11 2995 3271 3352 3456 3581 3660 3848 3926 3948 4169 4170 4429 5131 5186 5203 5615 5775 5998 6515 6550 7051 7402 8098 8316 8329 8450 Rev B 2006 33 Enzyme of cuts Positions c indicates the complementary strand Eael 15 9 63 1179 1568 1991 2758 3365 3916 4176 5519 5693 6084 6111 6336 7196 Eagl 5 2758 4176 5519 Eam1105l 1 7589 Earl 6 2305 c 2578 c 4443 c 5957 c 6167 c 6790 c Ecl136ll 1 728 Eclx 3 2758 4176 5519 Eco47I ll 1 1091 Eco57 12 1261 1305 c 1504 2107 c 2281 c 2662 c 3959 3988 5759 6191 6923 7935 c EcoO109 4 2739 3372 3454 6609 EcoRI 2 1843 3073 EcoRII 27 242 435 1151 1276 1388 1463 1517 1895 2033 2234 2357 2440 3271 3352 3456 3581 3660 3848 3926 3948 5131 5186 5203 5998 8316 8329 8450 Esp3 2 6552 6594 c Fnu4Hl 73 835 1267 1326 1348 1632 1639 1690 1693 1787 1879 1903 1942 1945 1991 2061 2210 2219 2255 2275 2290 2455 2458 2590 2716 2752 2827 2830 2981 3060 3160 3424 3693 3781 3784 3956 4005 4176 4179 4304 4486 4518 4532 4554 5025 5367 5522 5574 5585 5675 5680 5717 5758 5845 5848 5851 6087 6183 6224 6238 6339 6449 6558 6845 7074 7169 7196 7535 7863 8069 8072 8137 8280 8435 FnuDll 21 214 1436 1754 1851 4507 4531 4551 4927 5014 5679 5980 6012 6413 6493 6596 6598 6698 7030 7523 7853 8434 Fok 17 984 c 1135 c 15
20. AT3 is involved in providing nutritional support to the implanted blastocyst Experiments using mice in which STAT3 is ablated in a specific tissue or cell lineage have shown an involvement of STAT3 signalling in IL 6 dependent T cell proliferation 24 skin remodeling 25 deactivation of macrophages and neutrophils 26 motorneuron survival following axotomy 27 and mammary gland involution upon forced weaning 28 STAT3 signalling has recently been shown to be involved in leptin induced melanocortin production and body energy homeostasis 29 making it an attractive therapeutic target for the control of obesity Since the discovery that STAT3 is constitutively phosphorylated in v src transformed cells there is increasing evidence that aberrant STAT3 signalling is involved in oncogenesis In particular STAT3 is associated with cancers of the head neck and breast and with multiple myelomas leukemias and lymphomas 6 Thus the STAT3 signalling pathway is an attractive target for therapeutic intervention in a number of human cancers 1 2 EGFP STAT3 assay A cell screening assay examining the STAT3 signalling pathway Fig 1 2 has been developed The assay is based on Redistribution technology and quantifies the intracellular location of a EGFP STAT3 fusion protein in a stably transfected mammalian cell line The STAT3 Redistribution assay monitors translocation of EGFP STAT3 from the cytoplasm to the nucleus of cells stimulated wi
21. Al 3 1825 2306 2850 Bsgl 7 1236 c 1333 1657 2338 2566 2770 3145 c BsiEl 9 665 1100 2761 4179 4465 5522 7071 7220 8143 BsiHKAl 9 730 1717 3652 5726 5916 6424 6921 7006 8167 BsiV 22 203 1277 1440 1790 2166 2228 2229 2735 3287 3346 3762 4093 4516 4842 5327 5594 6138 6551 7999 8278 8444 8462 Bsll 22 203 1277 1440 1790 2166 2228 2229 2735 3287 3346 3762 4093 4516 4842 5327 5594 6138 6551 7999 8278 8444 8462 BsmAl 13 588 826 916 c 941 c 2396 c 3172 c 3211 c 3860 5464 6552 6594 c 6747 c 7523 BsmFl 12 329 480 648 2752 2986 3467 4014 c 5113 c 5185 c 5249 c 5764 6296 Bsml 3 3898 c 4240 4333 c Bsp1286l 17 730 1148 1277 1526 1717 3019 3652 4664 5559 5652 5726 5916 5978 6424 6921 7006 8167 BspDI 2 4425 6394 BspEI 3 1825 2306 2850 BspHl 3 6644 6749 7757 25 8010 38UM Chapter 11 Rev B 2006 32 Enzyme of cuts Positions c indicates the complementary strand BspMI 7 878 c 2120 c 4009 4028 5500 c 5881 6331 BspWI 53 137 244 366 398 437 530 554 803 1054 1199 1259 1272 1316 1325 1878 1911 1990 2215 2218 2280 2289 2451 2478 2520 2612 2757 2838 4464 4494 4526 4528 4570 4597 4627 5164 5236 5287 5366 5372 5604 5688 5711 5850 5856 5973 6009 6056 6323 6419 7471 7859 8431 8479 BsrBl 7 2826 3630 3636 4591 c 6226 c 6280 6747 c BsrDl 4 66 c 5846 7355 7529 c BsrFl 9 1095 1258 2756 2855 2875 4630 5932 6113 7504 BsrGl 2 97 1817 Bsrl 26 449 c 887 940 1034 c 1719 c 2430 c 2584 2653 2708 2883 c 3232 c 3440 3756 c 3866
22. GE Healthcare EGFP STAT3 Assay Product User Manual Codes 25 8010 38 25 8010 39 25 8010 40 25 8010 41 Page finder 5 4 7 Effect of different assay media 5 4 8 Effect of serum starvation 5 4 9 Effect of using DRAQ5 nuclear stain 5 4 10 Fixed Assay 5 4 11 AG490 imhibition curve 5 4 12 Results obtained on the IN Cell Analyzer 1000 6 Vector use details 6 1 General guidelines for vector use 6 2 Transient transfection with pCORON1000 EGFP STAT3 6 3 Stable cell line generation with pbCORON1000 EGFP STAT3 7 Quality control 7 1 EGFP STAT3 cell line 7 2 EGFP STAT3 expression vector 8 Troubleshooting guide 9 References 10 Related products 11 Appendix 11 1 Appendix A Restriction map of pCORON1000 EGFP STAT3 Front cover Top image BHK cells expressing the EGFP STAT3 fusion 22 22 23 23 23 24 25 25 25 25 26 26 26 27 28 30 31 31 1 Introduction 3 1 1 STAT3 3 1 2 EGFP STAT3 assay 4 2 Licensing considerations 6 2 1 Right to use 6 2 2 Legal 6 3 Product contents 8 3 1 Component summary 8 3 2 BHK derived cell line expressing EGFP STAT3 fusion protein NIF2027 8 3 2 1 BHK derived parental cell line 8 3 2 2 BHK derived EGFP STAT3 expressing cell line 8 3 3 EGFP STAT3 expression vector NIF2028 8 3 4 Materials and equipment required 9 3 5 IN Cell Analysis System 9 3 5 1 IN Cell Analyzer 3000 9 3 5 2 Nuclear Trafficking Analysis Module 9 3 5 3
23. IN Cell Analyzer 1000 9 3 6 EGFP STAT3 translocation assay on epifluorescence microscopes 10 3 7 Software requirements 10 4 Safety warnings handling and precautions 11 4 1 Safety warnings 11 4 2 Storage 12 4 3 Handling 12 4 3 1 Vector 12 4 3 2 Cells 12 5 Cell assay design 13 5 1 Culture and maintenance of BHK derived EGFP STAT3 expressing cell line 13 5 1 1 Tissue culture media and reagents required 13 5 1 2 Reagent preparation 13 5 1 3 Cell thawing procedure 14 5 1 4 Cell subculturing procedure 14 5 1 5 Cell seeding procedure 15 5 1 6 Cell freezing procedure 15 5 1 7 Growth characteristics 15 5 2 Assay set up 16 5 2 1 Live cell EGFP STAT3 assay using the IN Cell Analyzer 3000 16 5 2 2 Microplate set up for 96 well format assays 16 5 2 3 Schematic agonist assay protocol 16 5 2 4 Agonist assay protocol 96 well format 17 5 2 5 Fixed cell assay format 17 5 3 Results 18 5 3 1 Calculating the Z factor 18 5 3 2 Example results 18 5 4 Assay characterization 19 5 4 1 Translocation index 19 5 4 2 Summary of quantitative assay parameters 19 5 4 3 Seeding density 20 5 4 4 IL 6 dose response 20 5 4 5 Time course 20 5 4 6 Sensitivity of assay to DMSO ethanol and methanol 21 25 8010 38UM Pagefinder Rev B 2006 protein 25 minutes after the addition of control buffer only Bottom image BHK cells expressing the EGFP STAT3 fusion protein 25 minutes after stimulation with agonist 30 ng ml IL 6
24. Images shown are 1 14th of the actual image size acquired by the IN Cell Analyzer 3000 Biolmage is a Danish Biotech company specializing in developing drug candidates that exert their activity through modulation of protein translocation For more information visit their Web site at www bioimage dk 1 Introduction 1 1 STATS Signal transducers and activators of transcription STATs are a family of latent transcription factors that are activated in response to binding of polypeptide signalling molecules to cell membrane receptors Activated STAT proteins form dimers that translocate to the nucleus where they bind to promoter regions in target genes and ultimately modify the pattern of gene expression 1 2 3 4 Seven mammalian STAT genes have been identified in three chromosomal clusters 5 The encoded proteins STAT1 STAT2 STAT3 STAT4 STAT5a STAT5b and STAT6 are activated by distinct sets of cytokines and growth factors and each STAT protein activates a distinct set of target genes STAT proteins were initially described in the context of cell signalling where they contribute to such diverse processes as development differentiation proliferation and apoptosis 1 2 Increasingly a role for STAT proteins particularly STATS in transformation and tumor progression is being defined 6 STAT3 was first described as a DNA binding activity isolated from IL 6 stimulated hepatocytes that was capable of selectively binding to
25. M Alexa Fluor in 100 ul PBS For a DRAQ5 nuclear stained assay prepare an initial FF solution containing 1 0 ul 10 uM Oregon Green and 10 ul 10 uM Cy5 in 100 ul PBS Adjust these solutions if required Use 100 ul of FF solution for a 96 well plate and 40 ul FF solution for a 384 well plate 5 1 3 Cell thawing procedure Two cryo vials each containing 1 x 108 cells in 1 ml of Freeze medium are included with this assay kit The vials are stored frozen in the vapor phase of liquid nitrogen 1 Remove a cryo vial from storage 2 Holding the cryo vial dip the bottom three quarters of the cryo vial into a 37 C water bath and swirl gently for 1 2 minutes until the contents are thawed Do not thaw the cells for longer than 3 minutes as this decreases viability 3 Remove the cryo vial from the water bath and wipe it with 70 v v ethanol Transfer the cells immediately to a T 25 flask and add 5 ml pre warmed Growth medium drop wise to prevent cell damage Add a further 2 ml Growth medium and incubate at 37 C NOTE To ensure maximum cell viability do not allow the cells to thaw at room temperature and do not thaw the cells by hand 5 1 4 Cell subculturing procedure Incubation 5 CO gt 95 humidity 37 C The cells should be passaged at a ratio of 1 20 when they are 90 confluent 1 Warm all reagents to 37 C 2 Aspirate the medium from the cells and discard 3 Wash the cells with PBS Take care not to damage the cell laye
26. System The EGFP STAT3 assay has been developed and optimized for analysis using the IN Cell Analyzer 3000 in conjunction with the Nuclear Trafficking Analysis Module Please refer to the instrument user manual for details on instrument set up and the analysis module manual for details on the algorithm settings The assay can also be imaged and analyzed with the IN Cell Analyzer 1000 system For further information on either of these products please contact GE Healthcare 3 5 1 IN Cell Analyzer 3000 The IN Cell Analyzer 3000 is a line scanning laser based confocal imaging system with three high speed CCD cameras It has been developed specifically for performing information rich cellular assays very rapidly and at high resolution enabling high throughput and high content testing of drug compounds 3 5 2 Nuclear Trafficking Analysis Module The Nuclear Trafficking Analysis Module provides a method to quantify the movement of target molecules between the cytoplasm and the nucleus in either direction The fluorescence intensity of a target molecule in discrete nuclear and cytoplasmic regions is measured and the ratio of the sampled intensities calculated as an index of translocation 3 5 3 IN Cell Analyzer 1000 The IN Cell Analyzer 1000 is a bench top automated microscope system designed for imaging sub cellular end point assays The systems core components are a Nikon microscope xenon lamp and high resolution CCD camera Additional o
27. Tel 61 2 9899 0999 Fax 61 2 9899 7511 Austria Tel 01 57606 1619 Fax 01 57606 1627 Belgium Tel 0800 73 888 Fax 02 416 82 06 Canada Tel 1 800 463 5800 Fax 1 800 567 1008 Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1985 8327 Denmark Tel 45 16 2400 Fax 45 16 2424 Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 France Tel 01 6935 6700 Fax 01 6941 9677 http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK imagination at work Germany Tel 089 96281 660 Fax 089 96281 620 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 813 5331 9336 Fax 813 5331 9370 Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0800 8282821 Fax 0800 8282824 Norway Tel 815 65 555 Fax 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 Tel 7 495 956 5177 Fax 7 495 956 5176 South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Russia amp other C I S amp N I S Spain Tel 93 594 49 50 Fax 93 594 49 55 Sweden Tel 018 612 1900 Fax 018 612 1910 Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 USA Tel 1 800 526 3593 Fax 1 877 295 8102 25 8010 38UM Rev B 2006
28. actions An 18 amino acid flexible polypeptide chain coiled 25 8010 38UM Chapter 1 Rev B 2006 3 coil region links the oligomerization domain to the DNA binding domain which confers binding specificity The SH2 domain which is the most highly conserved STAT domain is necessary for receptor recruitment association with activating JAKs and STAT3 dimerization Finally the carboxy terminal transactivation domain is involved in transcriptional regulation of target genes In addition to phosphorylation within the dimerization domain at Tyr 5 phosphorylation of Ser 27 within the transactivation domain appears to contribute to maximal transcriptional activity of STAT3 possibly by enhancing the recruitment of transcriptional cofactors 20 21 Fig 1 1 Schematic showing major A Linker SE Y structural features of STAT3 including de gt a conserved tyrosine Y and serine S Oligomerization DNA binding Dimerization Transactivation phosphorylation sites STAT3 appears to regulate different genes in different cell types For example its induction of the anti apoptopic gene Bcl 2 in B cells results in cell proliferation while it s down regulation of c myc and c myb and induction of junB and IRF 1 in monocytic cells are linked to cell differentiation and growth arrest 22 Defining other functions for STAT3 has been somewhat difficult since STAT3 knockout mice die early in embryogenesis 23 it has been postulated that ST
29. actor 065 40 STAT3 OF1 0 242 1 16 0 29 1316 68 41 STATS DGI 0 27 116 031 12093 MOR 1 42 STAT3 0 H1 0 322 1 14 0 28 1463 78 43 STATS 0AQ 0 380 1 16 0 26 1579 86 S N 42 45 44 STATS 062 0 389 1 16 0 25 1285 98 45 STATS 0 C2 0 325 ii 0 27 1414 3 CY Unstim 257 46 STATS 0D2 0 344 115 02 1355 Stim 477 47 STATS OE2 it 312 1 12 0 26 1267 22 48 STAT3 OF2 0 304 109 023 1208 26 49 STAT3 062 0 320 14 0 25 1409 67 Tranclocation of EGFP STAT3on stimulation 50 STATS OH2 0 385 14 026 1318 91 with 30 ng ml IL 8 51 STAT3 0A3 D 347 1 13 0 27 1452 92 52 STATS 083 o 360 1 17 031 1518 34 an 53 STATS ojc3 0 334 1 15 0 32 1358 78 54 STAT3 O D3 D 359 1 15 0 29 1295 53 250 55 STAT3 0E3 0 389 115 054 1273 9 56 STAT3 OF3 0 365 1 12 0 3 1335 14 s 57 STATS 063 0 324 1417 027 138972 i 58 STATS 0 H3 0 286 1 11 0 24 1310 07 g D unstimulated 59 STAT3 DA 0 399 1 14 027 1236 29 3 5 m stimuieted 30 nami LS 60 STAT3 0B4 in 339 1 16 0 28 1267 83 61 STAT3 oc4 0 384 1 13 0 31 1342 65 1 00 62 STAT3 0b4 0 374 1 17 0 3 1282 14 B 63 STAT3 D E4 it 305 1 14 0 26 1281 27 0 50 64 STATS 0 F4 it 330 1 18 0 32 1476 88 65 STAT3 0 G4 0 339 1 15 0 27 1387 93 0 00 B6 STAT3 OHA D 38 08 0 26 1318 26 EE aa eee ee M4 X 100403_STAT3_smhPT6_Pop_002 z jg100403plate1_Population_002_v ig100403p2_Population_002_v001 9100403p3_Population_002_y001 va jaloa 4 Analysis of these results yielded a Z factor of 0 65 for the assay as performed using
30. c Stat3 deficient mice J Immuno 161 4652 4660 1998 Sano S et al Keratinocyte specific ablation of Stat3 exhibits impaired skin remodeling but does not affect skin morphogenesis EMBO 18 4657 4668 1999 Takeda K et al Enhanced Th1 activity and development of chronic entercolitis in mice devoid of Stat3 in macrophages and neutrophils Immunity 10 39 49 1999 Schweizer U et al Conditional gene ablation of Stat3 reveals differential signalling requirements for survival of motorneurons during development and after nerve injury in the adult J Cell Biol 156 287 297 2002 Chapman R S et al The role of Stat3 in apoptosis and mammary gland involution Conditional deletion of Stat3 Adv Exp Med Biol 480 129 38 2000 Bates S H et al Stat3 signalling is required for leptin regulation of energy balance but not reproduction Nature 421 856 859 2003 Macpherson I and Stoker M Polyoma transformation of hamster cell clones an investigation of genetic factors affecting cell competence Virology 16 147 151 1962 Macpherson l Characteristics of a hamster cell clone transformed by polyoma virus J Natl Cancer Inst 30 795 815 1963 Zhang J H et al A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays J Biomol Screen 4 67 73 1999 Freshney R I Cloning and Selection of Specific Cell Types in Culture of Animal Cells 3rd Ed
31. d 150 ul Assay medium Incubate at 37 C 5 CO for 30 minutes 5 Add 50 ul of the prepared four fold dilution stocks of the test and control compounds to the appropriate wells If Hoechst 33342 is used as the nuclear stain we recommend including a 6 second time interval between addition to each well The order of dispensing should be the same as the well imaging order This ensures that cells in each well have been stimulated for the same period of time prior to imaging The total well volume is 200 ul 6 After the first well has incubated for 25 minutes read the assay plate using the IN Cell Analyzer 3000 Alternatively for convenience the live cell assay could be fixed at the peak translocation time point and then imaged on the IN Cell Analyzer 3000 see section 5 2 5 for further details 7 Perform the data analysis using the Nuclear Trafficking Analysis Module A time interval of 6 seconds is required when using Hoechst 33342 as the nuclear stain This corresponds to the time taken to image one tile per well on the IN Cell Analyzer 3000 using 364 nm and 488 nm excitation on two excitation passes If DRAQS is used as the nuclear stain no delay is required since the 488 nm and 647 nm channels can be imaged simultaneously Any other changes to the protocol that may influence the time taken to image a well should be taken into consideration during test and control compound addition e g imaging more than one tile per well 5 2 5
32. ells per well E Control no IL 6 HB 30 ng ml IL 6 N 1 Nuc Cyt Translocation index 1 2000 4000 6000 8000 10000 12000 Cell Density cells well 5 4 4 IL 6 dose response Fig 5 6 shows an agonist dose response curve for the IL 6 response The data were collected 25 minutes after addition of agonist and demonstrate an ECso of 19 4 ng ml 34 N 1 Nuc Cyt Translocation index 1 0 T T T 2 1 0 2 3 4 log IL 6 ng ml 5 4 5 Time course Fig 5 7 shows a typical time course of the EGFP STAT3 translocation and indicates that the maximal translocation occurs approximately 25 minutes after stimulation with 30 ng ml IL 6 34 Control no IL 30 ng ml IL 6 N 1 Nuc Cyt Translocation index 0 10 20 30 40 50 Time minutes 25 8010 38UM Chapter 5 Rev B 2006 20 Fig 5 5 IL 6 induced EGFP STAT3 translocation as a function of seeding density Stimulated cells were treated with 30 ng ml IL 6 for 25 minutes prior to imaging Error SD n 8 replicates per data point Fig 5 6 IL 6 dose response curve using the supplied EGFP STATS cell line Error SD n 8 replicates per data point Fig 5 7 Time course of EGFP STAT3 translocation using 30 ng ml IL 6 as agonist Maximal response is seen after 25 minutes Error SD n 4 replicates per data point 5 4 6 Sensitivity of assay to DMSO ethanol and metha
33. er pending and foreign patent applications and Invitrogen IP Holdings Inc formerly Aurora Biosciences Corporation under US patents US 5625048 5777079 5804387 5968738 5994077 6054321 6066476 6077707 6090919 6124128 6319969 6403374 European patent 1104769 0804457 and Japanese patent JP 3283523 and other pending and foreign patent applications and Columbia University This product is sold under license from Columbia University under US patents 5491084 and 646826 Rights to use this product as configured are limited to internal use for screening development and discovery of therapeutic products NOT FOR DIAGNOSTIC USE OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rights are conveyed and University of Florida Research Foundation under patents US 5968750 5874304 5795737 6020192 and other pending and foreign patent applications and lowa Research Foundation The CMV promoter is covered under US patents 5168062 and 5385839 and its use is permitted for research purposes only Any other use of the CMV promoter requires a license from the University of lowa Research Foundation 214 Technology Innovation Center lowa City 1A452242 USA and Osaka University under US patents 5 719 042 5 844 082 CA patent 2145969 CN patent 1073157 and other pending and foreign patent applications The exact terms of use for the product as configured are specified in the Terms and Conditions of Sale accompanying the product but are limited to i
34. esuspend the cells with a 10 ml pipette until all the clumps have dispersed 7 Count the cells using either a CASY1 Cell Counter and Analyzer System Model TT or a hemocytometer 8 Using fresh Growth medium adjust the cell density to deliver the desired number of cells to each well For example to add 0 8 x 104 cells per well in a volume of 200 ul adjust the suspension to 4 x 104 cells per ml We recommend a concentration of 4 x 104 cells per ml 9 Dispense 200 ul of the cells into each well of the microplate except the well reserved for the FF solution see IN Cell Analyzer 3000 manual for further information 10 Optionally incubate the plates undisturbed on a level surface for 1 hour at room temperature approximately 20 C This treatment may reduce edge effects 11 Incubate the plated cells for 24 hours at 37 C 5 CO gt 95 humidity before starting the assay NOTE If the cells are near confluence prior to trypsinization they should be passaged into two T flasks They will then be ready for seeding the following day 5 1 6 Cell freezing procedure A decreased growth rate has been observed in cell populations after having undergone more than 10 passages It is recommended that the users make early freeze downs of cells at low passage number after expanding from passage 1 1 Harvest the cells as described in section 5 1 4 and resuspend the cells in a small volume of Growth medium 2 Count the cells as described
35. f the response and the kinetics of the translocation event achievable with different cell lines are unknown and may deviate considerably from the values specified in this manual 25 8010 38UM Chapter 6 Rev B 2006 25 7 Quality control 7 1 EGFP STAT3 cell line The EGFP STAT3 cell line is supplied at a concentration of 1 x 106 cells per ml in fetal calf serum containing 10 v v DMSO The cell line has the characteristics detailed in Table 7 1 Property Value Measurement method Assay stability Magnitude of response Quality Control Assay gt 0 9 Z factor 0 3 For 10 passages after dispatch Viability from frozen gt 80 CASY1 Cell Counter and Analyzer System Model TT Cell diameter um 18 21 CASY1 Cell Counter and Analyzer System Model TT Fluorescence at gt 40 000 for 10 FARCyte Gain 51 4 x 104 cells per passages after dispatch ml RFU 7 2 EGFP STAT3 expression vector The EGFP STAT3 expression vector is supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 9 at 250 ug ml The vector has the characteristics outlined in Table 7 2 Property Value Limits Measurement method Concentration 250 ug ml UV Absorbance 260 nm in water Purity Minimal A260 A2g09 ratio Between UV Vis Absorbance contamination of 1 8 2 2 260 nm and the DNA construct 280 nm by RNA or protein Expected restriction The restriction Agarose gel pattern digests should electrophoresis give fragments of the sizes shown
36. fter incubation in the absence or presence of 30 ng ml IL 6 The results shown in Fig 5 9 demonstrate that the assay tolerates a range of assay media Control no IL 6 ME 30 ng ml IL 6 Fig 5 9 The effect of different Assay Media on IL 6 induced translocation 34 of EGFP STAT3 Error SD n 4 replicates per data point x 2 5 ao pa O UO Sy 22 e 14 g 0 o o amp s x9 xs FG vg YT g Q XL LF YF GF Ce g tee FF FF eR EB os o Ff Fs S amp S SS f 2 Seer ee g Pe fe 2 eg es F Fs eee EE E N G Gg x Y Ra GG Ly T T os oT Sos S 2 2 Ss gt e F GV gs F RS So amp o F amp my my o amp my 9 S S Q i S S a o Vow LS S a a a F se 2 amp amp vu ey ul GG Ra ww amp S a ee 2222 F T Assay medium formulation 5 4 8 Effect of serum starvation To determine the effect of serum starving the cells prior to the assay cells were incubated in Assay medium for 1 4 hours The results shown in Fig 5 10 demonstrate that the assay tolerates a range of serum starvation times including the 25 minutes IL 6 stimulation time in the recommended Assay medium E Control no IL 6 3 ME 30 ng ml IL 6 Fig 5 10 The effect of serum starvation on IL 6 induced translocation of EGFP STATS3 Error v SD n 4 replicates per data point E 2 so 5 o D oz a 5 H 0 1 2 3 4 Starvation time hours
37. further information refer to the material safety data sheet s and or safety statementis 4 2 Storage The EGFP STAT3 expressing DNA construct NIF2028 should be stored at 15 C to 30 C The BHK derived cells expressing the EGFP STAT3 fusion protein NIF2027 should be stored at 196 C in liquid nitrogen 4 3 Handling Upon receipt the cells should be removed from the cryo porter and transferred to a gaseous phase liquid nitrogen storage unit Care should be taken to ensure that the cells are not warmed unless they are required immediately The vector should be removed from the cryo porter and stored at 20 C until required 4 3 1 Vector After thawing the DNA sample centrifuge briefly to recover the contents 4 3 2 Cells Do not centrifuge the cell samples upon thawing 25 8010 38UM Chapter 4 Rev B 2006 12 5 Cell assay design 5 1 Culture and maintenance of BHK derived EGFP STAT3 expressing cell line 5 1 1 Tissue culture media and reagents required The following media and buffers are required to culture maintain and prepare the cells and to perform the assay e GIBCO Dulbecco s Modified Eagle Media DMEM with Glutamax 1 Invitrogen life technologies 31966 021 or equivalent e GIBCO Nutrient Mixture F 12 Ham medium with Glutamax Invitrogen life technologies 31765 027 or equivalent e Fetal Bovine Serum FBS JRH Biosciences 12103 or equivalent e GIBCO Penicillin Streptomycin P S 10 000 units ml
38. here o standard deviation u mean signal C positive control c negative control Lea 5 3 2 Example results The following figures Fig 5 3 and Fig 5 4 are taken from a single experiment providing an example of the image quality and results that can be obtained with the EGFP STAT3 assay using the IN Cell Analyzer 3000 Fig 5 3 shows images acquired on the IN Cell Analyzer 3000 using the supplied BHK derived EGFP STAT3 cell line Images shown here are 1 14th of the full images acquired by the system Fig 5 3 The BHK derived EGFP STAT3 expressing cells 25 minutes after stimulation with A control buffer only or B 30 ng ml IL 6 The field of cells shown is not the same The nuclear blue image channel is not displayed here Fe Fie Edit view Insert Format Tools Data SAR Window Help D Sn GRY SBR o Belz mA i B 0 Q arial 0 B 7u ESSER ou 88 ae Fig 5 4 Data from the example i z experiment generated by the Nuclear E eo ga i 34 Plate Cycle Well Misg Cells Nuc Cyt Std Dev Nuc Intsty unstimulated stimulated 30 ng ml IL 5 Trafficking Analysis Module exported 35 STAT3 0 A1 it 322 1 09 0 26 1182 95 Mean 114 2 39 36 STATS 081 o 298 1 12 0 26 1256 53 SD 0 03 0 11 to and analyzed in Microsoft Excel 37 STAT3 0 c1 0 347 1 4 0 25 1201 01 n 48 48 38 STATS 0D1 0 340 1 09 0 27 1135 87 39 STATS Oct 0 75 145 025 1457 71 Z F
39. in Table 7 3 Enzymels of cuts Fragment s size bp BamHI 2 3415 5078 BsrGl 2 1720 6773 Hindlll 2 3815 4678 Notl 1 8493 Pvul 3 1938 2755 3800 25 8010 38UM Chapter 7 Rev B 2006 26 Table 7 1 Quality control information for EGFP STAT3 cell line Table 7 2 Quality control information for the EGFP STAT3 expression vector Table 7 3 Expected restriction pattern for the EGFP STAT3 expression vector 8 Troubleshooting guide Problem 1 Low assay response positive vs negative controls 2 Low nuclear intensity 3 Image is out of focus 4 Cells do not adhere to well bottom in plate 5 Shading across image field 25 8010 38UM Chapter 8 Rev B 2006 Possible cause 1 1 Passage number too high 1 2 Cell density too low or too high 1 3 Incorrect selection of analysis parameters 1 4 Incorrect assay incubation conditions 1 5 Reagents were not stored properly or they are out of date 1 6 Cells have been stressed during assay 2 1 Nuclear stain concentration too low 2 2 Nuclear stain incubation time too short 3 1 Autofocus Offset is chosen incorrectly or the system may need to be realigned 4 1 Plating density too high 5 1 Flat field correction not applied or flat field solution too weak ar Remedy 1 1 Start a fresh batch of cells from an earlier passage number Cells should be expanded and additional vials should be frozen down from the vials delivered w
40. ith the kit 1 2 Verify density of cell plating adjust plating density to values that yield optimal assay response 1 3 Check that the primary parameters are correct and suitable for the cells currently in use 1 4 Ensure that proper incubation is maintained as consistently as possible during the assay When plates are out of the CO incubator for extended periods it is essential that HEPES buffer be added to the medium to maintain the correct pH 1 5 Repeat assay with fresh reagents 1 6 Use actively growing cells maintained at 37 C Pre warm reagents to 37 C 2 1 Adjust Nuclear stain concentration to recommended level 2 2 Adjust Nuclear stain incubation time to recommended length 3 1 Alignment and calibration of instrument Perform Z stack on cells Change Autofocus Offset 4 1 Reduce plating density 5 1 Apply flat field correction or adjust flat field solution 9 References 10 11 12 13 14 15 16 17 18 19 20 21 22 Schindler C et al Transcriptional responses to polypeptide ligands the JAK STAT pathway Annu Rev Biochem 64 621 651 1995 Darnell J E Stats and gene regulation Science 277 1630 1635 1997 Kisseleva T et al Signalling through the JAK STAT pathway recent advances and future challenges Gene 285 1 24 2002 Levy D E et al What does Stat3 do J Clin Invest 109 1143 1148 2002 Copeland N G et a
41. ition Wiley Liss Inc Chapter 11 pp 161 178 1994 25 8010 38UM Chapter 9 Rev B 2006 29 10 Related products Product Name Code GFP Assays GFP PLC8 PH domain assay See below GFP Rac1 assay See below GFP MAPKAP k2 assay See below AKT1 EGFP assay See below EGFP 2xFYVE assay See below EGFP SMAD2 assay See below EGFP NFATcl assay See below CypHer pCORON1000 VSV G tag Expression vector 25 8008 51 pCORON1000 SP VSV G tag Expression vector 25 8009 92 CypHer5E labeled anti VSV G antibody PA45407 CypHer5E NHS ester 1 mg pack PA15401 CypHer5E NHS ester 5 mg pack PA15405 Use of the GFP assays is limited as stated in the Terms and Conditions of Sale The product codes vary accordingly Please contact your local representative for details IN Cell Analysis system N Cell Analyzer 3000 25 8010 11 Nuclear Trafficking Analysis Module for the N Cell Analyzer 3000 63 0048 96 N Cell Analyzer 1000 25 8010 26 Nuclear Trafficking Analysis Module for the N Cell Analyzer 1000 25 8010 31 25 8010 38UM Chapter 10 Rev B 2006 30 11 Appendix 11 1 Appendix A Restriction map of pPCORON1000 EGFP STAT3 The following enzymes do not cut the vector Apal Ascl BbrPI Bpu1102I BsiWI Bsp1201 Bst11071 BstEll Celll EcoNI EcoRV Espl Kspl Nrul Pacl PmaCl Pmel Pmil Sacll SgrAl Swal Enzyme of cuts Positions c indicates the complementary strand Aatl 2 3200 5418 Aatll 5 279
42. l Evolution of the mammalian Stat gene family Genomics 29 225 228 1995 Bowman T et al Stats in oncogensis Oncogene 19 2472 2488 2000 Akira S et al Molecular Cloning of APRF a novel INF stimulated gene factor 3 p91 related transcription factor involved in the go130 mediated signalling pathway Cell 77 63 71 1994 Zhong Z et al Stat3 and Stat4 Members of the family of signal transducers and activators of transcription Proc Natl Acad Sci 91 4806 4810 1994 Hirano T Signaling mechanisms through gp130 a model of the cytokine system Cytokine and Growth Factor Reviews 8 241 252 1997 Boccaccio C et al Induction of epithelial tubules by growth factor HGF depends on the STAT pathway Nature 15 285 288 1998 Bild A H et al Cytoplasmic transport of Stat3 by receptor mediated endocytosis EMBO 21 3255 3263 2002 Bhattacharya S et al Regulation of Stat3 nuclear export J Clin Invest 111 553 559 2003 Callus B A et al Interleukin 3 induced activation of the JAK STAT pathway is prolonged by proteasome inhibitors Blood 91 3182 3192 1998 Hilton D J Negative regulators of cytokine signal transduction Cell Mol Life Sci 55 1568 1577 1999 Irie Sasaki J et al CD45 is a JAK phosphatase and negatively regulates cytokine receptor signalling Nature 409 349 354 2001 Myers M P et al TYK2 and JAK2 are substrates of protein tyrosine phosphatase 1B J Bi
43. monly referred to as a GAS element IFN g activated sequence and initiate a change in transcription 2 In mammalian cells the time scale for STAT activation and nuclear import is typically in the range of 5 60 minutes following stimulation During the subsequent period of signalling decay STATs are exported from the nucleus back to the cytoplasm in the case of STAT3 this event is dependent on multiple nuclear export signals 12 As would be expected STAT signalling is highly regulated and several regulatory processes are involved although the complete pathway for individual STATs remains unclear Deactivation of signalling from the cell surface can occur by degradation of the receptor ligand complex via the ubiquitin proteosome pathway 13 and through the induction of the suppressor of cytokine signaling SOCS family of proteins which inhibit Jaks 14 Further a number of phosphatases acting either at the receptor or at the receptor associated Jak can lead to a cessation of STAT signalling 15 16 The STAT proteins themselves can also be dephosphorylated by specific phosphatases in both the cytoplasmic and nuclear compartments 17 18 Protein inhibitors of activated STATs PIAS bind to STAT dimers in the nucleus preventing DNA recognition 19 Structurally STAT3 is similar to other STAT proteins and has four main domains Fig 1 1 The first an amino terminal oligomerization domain is involved in stabilizing STAT3 dimer DNA inter
44. nol The EGFP STAT3 translocation was measured in the presence of DMSO lt 2 ethanol lt 2 or methanol lt 2 As can be seen in Fig 5 8 the IL 6 induced translocation can withstand at least 0 5 of each solvent 5 Control no IL 6 34 EE 30 ng ml IL 6 x oO 2 p 5S Bx D Q o D oz e 14 9 0 00 0 10 0 25 0 50 1 00 2 00 DMSO E Control no IL 6 3 EE 30 ng ml IL 6 x oO g 2 p 5 pa O Q 53 n I T T Cc 1 4 2 kK 0 0 00 0 10 0 25 0 50 1 00 2 00 Ethanol 5 Control no IL 6 34 E 30 ng ml IL 6 x 5 S 24 5 ins pa D VO o D 22 I 14 S kK 0 00 0 10 0 25 0 50 1 00 2 00 Methanol 25 8010 38UM Chapter 5 Rev B 2006 21 Fig 5 8a Effect of DMSO on the IL 6 induced EGFP STAT3 translocation Error SD n 8 replicates per data point Fig 5 8b Effect of ethanol on the IL 6 induced EGFP STAT3 translocation Error SD n 8 replicates per data point Fig 5 8c Effect of methanol on the IL 6 induced EGFP STAT3 translocation Error SD n 8 replicates per data point 5 4 7 Effect of different assay media To determine the effect of varying the assay medium on the IL 6 induced EGFP STATS translocation cells were assayed in either Nutrient Mixture F 12 Ham medium Ham F 12 or Dulbecco s Modified Eagle medium DMEM with a range of additives 10 mM HEPES BSA and FBS The results were collected 25 minutes a
45. nternal use for development and discovery of therapeutic products No rights other than those expressly granted are conveyed 2 2 Legal GE and GE monogram are trademarks of General Electric Company Cy is a trademark of GE Healthcare companies Biolmage and Redistribution are trademarks of Biolmage A S Biocarta is a trademark of Biocarta Inc FuGENE is a trademark of Fugent LLC Microsoft is a trademark of Microsoft Corporation 25 8010 38UM Chapter 2 Rev B 2006 6 Hoechst is a trademark of Aventis Geneticin is a trademark of Life Technologies Inc DRAQS is a trademark of Biostatus Limited 2006 General Electric Company All rights reserved GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specification and features shown herein or discontinue the product described at any time without notice or obligation Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK 25 8010 38UM Chapter 2 Rev B 2006 7 3 Product contents 3 1 Component summary e BHK derived cells expressing the EGFP STAT3 fusion protein two vials each containing 1 ml and 1 x 108 cells NIF2027 e pCORON1000 EGFP STAT3 expression vector one vial containing 10 ug DNA at a concentration of 250 ug ml s
46. ol Chem 276 4771 47774 2001 Yamamoto T et al The nuclear isoform of protein tyrosine phosphatase TC PTP regulates interleukin 6 mediated signalling pathway through Stat3 dephosphorylation Biochem Biophys Res Comm 297 811 817 2002 Hoeve J et al Identification of a nuclear Stat1 protein tyrosine phosphatase Mol Cell Biol 22 5662 5668 2002 Chung C D Specific inhibition of Stat3 signal transduction by PIAS3 Science 278 1803 1805 1997 Wen Z et al Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation Cell 82 241 250 1995 Wen Z et al Mapping of Stat3 serine phosphorylation to a single residue 727 and evidence that serine phosphorylation has no influence on DNA binding of Stat1 and Stat3 Nucleic Acids Res 25 2062 2067 1997 Hirano T et al Roles of Stat3 in mediating the cell growth differentiation and survival signals relayed through the IL 6 family of cytokine receptors Oncogene 19 2548 2556 2000 25 8010 38UM Chapter 9 Rev B 2006 28 23 24 25 26 27 28 29 30 31 32 33 Takeda K et al Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality Proc Natl Acad Sci 94 3801 3084 1997 Takeda K et al Stat3 activation is responsible for IL 6 dependent T cell proliferation through preventing apoptosis Generation and characterisation of T cell specifi
47. optimized for agonist format see sections 5 2 3 and 5 2 4 It is essential that the number of cells per well in the assay plates be consistent in order to minimize assay variability IL 6 is used as a reference agonist with a typical ECs value of 19 4 ng ml The EGFP STAT3 assay can be used with either Hoechst or DRAQ5 as the Nuclear stain The majority of data presented in this manual were obtained using Hoechst stain As explained in the IN Cell Analyzer 3000 user manual each run must contain a flat field well to compensate for variations in fluorescence intensity across each image It is possible to prepare a plate solely for this purpose Alternatively a designated well on each plate can contain flat field solution When seeding the plate this well must not contain any cells if the auxiliary flat field correction tool is to be applied in the analysis module 5 2 3 Schematic agonist assay protocol Fig 5 2 shows a typical schematic of the agonist assay The cells should be seeded in the appropriate microplate the day before the experiment The Growth medium is decanted the cells are washed and Assay medium added to each well Following a 30 minute incubation at 37 C 5 CO3 controls and test compound are added to required wells After 25 minutes incubation the microplates are START Fig 5 2 Flow diagram showing a basic protocol suitable for a EGFP STAT3 ae agonist screen y Incubate overnight 37 C 5 CO2
48. penicillin G sodium and 10 000 ug ml streptomycin sulfate Invitrogen life technologies 15140 122 or equivalent e Geneticin G418 Sigma G 7034 or equivalent e GIBCO Trypsin EDTA in HBSS w o calcium or magnesium Invitrogen life technologies 25300 054 or equivalent e GIBCO HEPES Buffer 1M solution Invitrogen life technologies 15630 056 or equivalent e Bovine serum albumin BSA Sigma A 7888 or equivalent e GIBCO Phosphate Buffered Saline PBS Dulbecco s w o calcium magnesium or sodium bicarbonate Invitrogen life technologies 14190 094 or equivalent e Dimethylsulfoxide DMSO Sigma D 2650 or equivalent e Hoechst 33342 Molecular Probes H 21492 or similar e DRAQ5 Biostatus e Interleukin 6 Human Recombinant E coli IL 6 Calbiochem 407652 e Cy 5 monocarboxyl dye GE Healthcare PAO5111 or equivalent e Oregon Green 2 7 difluorofluorescein Molecular Probes D 6145 e Alexa Fluor carboxylic acid succinimidy ester Molecular Probes A 10168 e Formalin solution 10 neutral buffered 4 w v Formaldehyde Sigma HT50 1 2 e Phosphate buffered saline tablets Sigma P 4417 e Standard tissue culture plastic ware including tissue culture treated flasks T flasks centrifuge tubes and cryo vials 5 1 2 Reagent preparation NOTE the following reagents are required but not supplied e Heat inactivation of FBS 30 minutes in water bath at 56 C e Growth medium DMEM with Glutamax 1 supplemented with
49. ptional modules include liquid handling both compound and reservoir dispense as well as aspirate and temperature control to enable imaging live cell assays over extended periods and in real time The IN Cell Analyzer 1000 system has a number of complementary analysis modules as well as the capability to export images and data into other commercial analysis packages The Nuclear Trafficking Analysis Module for the IN Cell Analyzer 1000 measures nuclear translocation by comparing sampled fluorescence intensity of the labeled molecule in the nuclear and cytoplasmic compartments 25 8010 38UM Chapter 3 Rev B 2006 9 3 6 EGFP STAT3 translocation assay on epifluorescence microscopes For speed of screening and quality of the images obtained we recommend performing the EGFP STAT3 assay on the IN Cell Analyzer systems However it is possible to adapt the assay to be read on alternative imaging platforms Laboratory grade inverted epifluorescence microscopes such as the Nikon Diaphot or Eclipse models or the Zeiss Axiovert model are suitable for image acquisition A high quality objective Plan Fluor 40 x 1 3 NA or similar and epifluoresence filter sets compatible with GFP and the desired nuclear dye will be required A motorized stage with multi well plate holder and a heated stage enclosure are also recommended for assays performed on epifluorescence microscopes and a suitable software package will be required for image analysis 3 7 Soft
50. r while washing but ensure that the entire cell surface is washed 4 Aspirate the PBS from the cells and discard 5 Add Trypsin EDTA 2 ml for T 75 flasks and 4 ml for T 162 flasks ensuring that all cells are in contact with the solution Wait for 3 10 minutes for the cells to round up loosen Check on an inverted microscope 6 When the cells are loose tap the flask gently to dislodge the cells Add Growth medium 8 ml for T 75 and 6 ml for T 162 flasks and gently resuspend the cells with a 10 ml pipette until all the clumps have dispersed 7 Aspirate the cell suspension and dispense 0 5 ml cells into a new culture vessel 25 8010 38UM Chapter 5 Rev B 2006 14 5 1 5 Cell seeding procedure The following procedure is optimized for cells grown in standard T 75 and T 162 flasks to be seeded into 96 well microplates 1 Warm all reagents to 37 C 2 Aspirate the medium from the cells and discard 3 Wash the cells with PBS Take care not to damage the cell layer while washing but ensure that the entire cell surface is washed 4 Aspirate the PBS from the cells and discard 5 Add Trypsin EDTA 2 ml for T 75 and 4 ml for T 162 flasks ensuring that all cells are in contact with the solution Wait for 3 10 minutes for the cells to round up loosen Check on an inverted microscope 6 When the cells are loose tap the flask gently to dislodge the cells Add Growth medium 3 ml for T 75 and 6 ml for T 162 flasks and gently r
51. ry strand Hphl 20 530 1122 1125 c 1455 1479 1608 2274 3162 c 3184 c 3252 4084 4735 5791 c 6569 c 6578 c 6862 c 6897 7103 c 7519 7746 Ital 73 835 1267 1326 1348 1632 1639 1690 1693 1787 1879 1903 1942 1945 1991 2061 2210 2219 2255 2275 2290 2455 2458 2590 2716 2752 2827 2830 2981 3060 3160 3424 3693 3781 3784 3956 4005 4176 4179 4304 4486 4518 4532 4554 5025 5367 5522 5574 5585 5675 5680 5717 5758 5845 5848 5851 6087 6183 6224 6238 6339 6449 6558 6845 7074 7169 7196 7535 7863 8069 8072 8137 8280 8435 Kasl 1 5612 Kpni 2 1896 5073 Ksp632l1 6 2305 c 2578 c 4443 c 5957 c 6167 c 6790 c Mael 15 154 753 1058 1087 2326 2632 2641 3214 4231 4582 5420 5474 7396 7731 7984 Maell 23 75 276 288 329 412 493 598 1172 1385 1556 2543 2656 2975 4624 4734 4777 4789 5729 5916 6667 6987 7360 7776 Maelll 27 215 302 651 839 902 1290 1779 1921 2262 2436 3147 3708 3987 4289 4545 4557 5733 6039 6540 6928 7116 7269 7327 7658 7941 8057 8120 Maml 2 4421 6406 Mbol 40 662 747 1607 1755 1793 1834 2322 2412 2634 2992 3167 3242 3357 3482 3752 3827 4422 4426 4462 5016 5784 5862 5943 5952 6030 6407 6906 6942 6959 7217 7263 7281 7622 7727 7739 7817 7825 7836 7911 8489 Mboll 26 967 1350 c 1395 c 1398 c 1593 2113 c 2117 2197 2322 2460 2595 3055 3981 4460 4596 c 5436 c 5974 6184 6264 c 6807 6916 6994 7749 7820c 7972 c 8B484 c Mcrl 9 665 1100 2761 4179 4465 5522 7071 7220 8143 Mfel 2 3344 4328 Mlul 1 1849 MIuNI 3 11 65 5695 Mnll 61 703 c 870 c 1116 c
52. ter 5 Rev B 2006 19 Table 5 1 Results from a typical single assay performed using the suggested protocol Signal to noise is calculated as mean signal mean background background standard deviation 32 Magnitude of response is calculated as mean signal mean background CV is calculated as standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of assay quality 32 It is defined in section 5 3 1 Table 5 2 Summary results from assays performed by different operators on different occasions using the suggested protocol SD shown is the Standard deviation of the assays Signal to noise is calculated as mean signal mean background background standard deviation 32 Magnitude of response is calculated as mean signal mean background CV is calculated as standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of assay quality 32 It is defined in section 5 3 1 5 4 3 Seeding density Fig 5 5 shows the effect of varying seeding density when the assay is performed in a 96 well microplate The data were collected 25 minutes after the addition of assay buffer control or 30 ng ml IL 6 Significant differences between stimulated 30 ng ml IL 6 and non stimulated no IL 6 cells were seen at cell densities ranging from 0 2 x 104 to 1 2 x 104 cells per well We recommend seeding the cells at a density of 0 8 x 104 c
53. th IL 6 Fig 1 2 Schematic of the STAT3 Cytokines signalling pathway provided with y permission from BioCarta www biocarta com DNA P Transcription P Ser7 P LSRE GAS 25 8010 38UM Chapter 1 Rev B 2006 4 Fig 1 3 This assay is optimized for image acquisition and analysis on the IN Cell Analyzer 3000 using the Nuclear Trafficking Analysis Module although the assay can also be imaged on the IN Cell Analyzer 1000 and other subcellular imaging systems Using the recommended assay format IL 6 has an ECs value of 19 4 ng ml Fig 1 3 Agonist induced redistribution of EGFP STAT3 from the cytoplasm to Agonist 35 minutes the nucleus Un stimulated cell Stimulated cell Majority of EGFP STAT3 EGFP STAT3 localized in cytoplasm redistributes to nucleus 25 8010 38UM Chapter 1 Rev B 2006 5 2 Licensing considerations 2 1 Right to use Use of this assay is limited as stated in the terms and conditions of sale These vary in accordance with the product code purchased Description Product Code EGFP STAT3 Assay Screening applications 25 8010 38 EGFP STAT3 Assay Research applications 25 8010 39 EGFP STAT3 Assay 6 month assay evaluation 25 8010 40 EGFP STAT3 Assay 12 month assay evaluation 25 8010 41 The assay was developed in collaboration with Biolmage A S and sold under license from Biolmage A S under patents US 6172188 US 5958713 US6518021 EP851874 EP0815257 EP0986753 and oth
54. tion map is shown in chapter 11 appendix A Bsr GI 97 Fig 3 1 Vector map of the supplied My ne EGFP STAT3 expression vector Pvul 665 CMV promoter Hind 111 757 Chimeric intron Ampicillin resistancegene Pvul 7220 BamHI 6407 ___ pCORON1000 EGFP STAT3 8493 bp Synthetic poly A Neomycin resistance gene Hin dill 5435 SV40 minimum origin of replication SV40 enhancer early promoter flori Pvul 4465 SS Noti 4176 SV40 late polyA 25 8010 38UM Chapter 3 Rev B 2006 8 3 4 Materials and equipment required The following materials and equipment are required but not provided e Microplates For analysis using the IN Cell Analyzer 3000 Packard Black 96 Well ViewPlates Packard Cat 6005182 should be used For assays in 384 well format please e mail incellanalyzer uk amersham com for recommendations e ACASY 1 Cell Counter and Analyzer System Model TT Sch rfe System GmbH is recommended to ensure accurate cell counting prior to seeding Alternatively a hemocytometer may be used e Environmentally controlled incubator 5 CO gt 95 relative humidity 37 C e Imager microscope e g IN Cell Analyzer 3000 e Laminar flow cell culture bench e Tissue culture flasks T flasks and pipettes e Controlled freezing rate device providing a controlled freezing rate of 1 C per minute e Standard tissue culture reagents and facilities section 5 1 1 3 5 IN Cell Analysis
55. upplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 0 NIF2028 e User manual 3 2 BHK derived cell line expressing EGFP STAT3 fusion protein NIF2027 3 2 1 BHK derived parental cell line The parental cell line BHK 21 C 13 ATCC CCL 10 was derived from the kidneys of five unsexed one day old hamsters 30 Following 84 days of continuous cultivation interrupted only by an eight day preservation by freezing clone 13 was initiated by single cell isolation 31 3 2 2 BHK derived EGFP STAT3 expressing cell line BHK 21 cells were transfected with the pCORON1000 EGFP STAT3 vector supplied using the FUGENE 6 transfection method according to the manufacturer s instructions A stable clone expressing the recombinant fusion protein was selected using 1 mg ml Geneticin for approximately two weeks The isolated clone was chosen at passage 12 and grown for a further 10 passages before being sorted using a FACS machine Following sorting the cells were grown for a further 8 passages before freezing The cells have been tested and found to be negative for mycoplasma bacteria and yeast testing details available on request 3 3 EGFP STAT3 expression vector NIF2028 The 8 493 kb plasmid PCORON1000 EGFP STAT3 contains a bacterial ampicillin resistance gene and a mammalian neomycin resistance gene see Fig 3 1 The sequence of the construct is available on a CD upon request Please e mail incellanalyzer amersham com A detailed restric
56. uts Positions c indicates the complementary strand Mwol cont d 5164 5236 5287 5366 5372 5604 5688 5711 5850 5856 5973 6009 6056 6323 6419 7471 7859 8431 8479 Nael 2 4632 6115 Narl 1 5613 Neil 15 1137 1791 2160 2172 2997 4171 4172 4431 5617 5777 6517 6552 7053 7404 8100 Ncol 5 514 1106 5030 5326 6045 Ndel 2 388 1986 Nadell 40 662 747 1607 1755 1793 1834 2322 2412 2634 2992 3167 3242 3357 3482 3752 3827 4422 4426 4462 5016 5784 5862 5943 5952 6030 6407 6906 6942 6959 7217 7263 7281 7622 7727 7739 7817 7825 7836 7911 8489 NgoMl 2 4630 6113 Nhel 1 1086 Nlalll 43 118 136 458 518 1110 1344 1374 1569 1764 1809 2011 2409 2841 2847 3042 3300 3491 3516 3612 3798 3819 3834 4128 4161 4196 5034 5167 5239 5330 5487 5832 6018 6049 6075 6564 6648 6753 7146 7182 7260 7270 7761 8481 NlalV 33 621979 1145 1683 1894 1958 2176 2233 2740 2741 2860 2994 3118 3351 3455 3666 3767 4663 4675 4696 5071 5137 5209 5614 5649 6409 6702 7292 7503 7544 7638 8410 8449 Notl 1 4176 Nsil 3 2849 5169 5241 Nspl 9 2011 2409 2841 3300 3798 5167 5239 6018 6564 NspV 1 6295 PaeR7I 1 1838 PfIMI 2 3287 3762 PinAl 1 1095 Plel 16 558 c 836 c 952 c 1068 c 1823 c 1979 2082 2402 2959 c 4116 4171 4791 4799 c 6278 c 7598 8101 c Ppu10l 5 2845 5165 5237 PpuMlI 3 2739 3372 3454 Psp1406 2 6987 7360 Pstl 6 839 2204 2211 3072 4006 5666 Pvul 3 665 4465 7220 Pvull 5 2460 3426 3786 5095 5719 Real 5 6644 6749 7757 Rsal 18 99 373 453 486 537 702 1064 1537 1819 1894
57. ware requirements IN Cell Analysis System Nuclear Trafficking Analysis Modules are available from GE Healthcare for automated image analysis of the EGFP STAT3 assay for both the IN Cell Analyzer 1000 and the IN Cell Analyzer 3000 Analyzed data are exported as numerical files in ASCII format ASCII format data can be imported into Microsoft Excel Microsoft Access or any similar package for further data analysis as desired Confocal or epifluorescence microscope Suitable software will be required for analysis of images acquired on microscopes other than the IN Cell Analysis systems 25 8010 38UM Chapter 3 Rev B 2006 10 4 Safety warnings handling and precautions 4 1 Safety warnings Warning For research use only Not recommended or intended for diagnosis of disease in humans or animals Do not use internally or externally in humans or animals All chemicals should be considered as potentially hazardous We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Care should be taken to avoid contact with skin or eyes In the case of contact with skin or eyes wash immediately with water CAUTION Contains genetically modified material Genetically modified cells supplied in this package are for use

25-8010-38UM Rev B 2006.indd - GE Healthcare Life Sciences

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