GFP-PLCδ-PH domain Assay - GE Healthcare Life Sciences
Contents
1. s Chapter 2 Licensing Considerations 2 1 Product right to use 2 2 Rex xc i Chapter 3 Product Contents 3 1 Components summary 3 2 CHO derived cell line expressing GFP PLC5 PH domain fusion protein NIF1954 3 2 1 CHO derived parental cell line _ 3 2 2 CHO derived GFP PLC PH domain expressing cell line 3 3 GFP PLC PH domain expression vector NIF1991 3 4 Materials and equipment required 3 5 Instrument requirements 3 5 IN Cell Analyzer ix acumen icc cm mn mn ncn 3 5 1 Analysis of GFP PLC6 PH domain Assay on epifluorescence HIIGFOSOODSI a n E 3 6 Software requirements Chapter 4 Safety Warnings Handling and Precautions 4 1 Safety wamlngS DER Ras RR EE 4 2 Hur Me 4 3 HANGING iuum IER os VECO qr 4 3 2 CUS aos si Chapter 5 Cell Assay Design 5 1 Culture and maintenance of CHO derived GFP PLC5 PH domain expressing cell line Tissue culture media and reagents required2. Reagent x2 122 2215 6 awas eee ee k Hi n Cell thawing Procedures uuu setae ees eee xc pum Cell seeding procedure 5 1 1 5 1 2 5 1 3 5 1 4 Cell sub culturing procedure 5 1 5 5 1 6 Cell freezing 5 1 7 Growth CharactenStiGS sores v n aim ntt a RR Ra RR sn n 25 8007 26UM Page finder Rev A 2003 3 2 Assayseti 22 cee eee m w sma nnm 33 wa 5 2 1 Live cell GFP PLC PH domain assay using the IN Cell Analyzer 3000 i nuin nma m a Eam RR Rn 5 2 2 Microplate set up for 96 well format assays 5 2 3 Schematic antagonist assay protocol for use with the IN Cell Analysis 5 5 5 2 4 Detailed antagonist assay protocol 96 well format 5 2 5 Jimingscledule REIR x E ISTE EE 5 2 6 Important considerations ss ra hmmm so e 5 3 RESUS oe c 5 3 1 Calculating Z factor c yaaa E ese 5 3 2 Example TestltS z s m teni em Ir ne ayia 5 4 Assay characterization 5 41 Trarnslocation index x sex etn ex rmx RR nme Rana 5 4 2 Summary of quantitative assay parameters 54 3 Seedingdensily iiis dett kr eed eee RR n sm a w NE xn 5 4
3. 17 35 10 24 19 39 12 835 1776 1892 1903 2113 2201 2321 2389 2392 2517 2699 2731 2745 2767 3238 3580 3735 3787 3798 3888 3893 3930 3971 4058 4061 4064 4300 4396 4437 4451 4552 4662 4771 5058 5287 5382 5409 5748 6076 6282 6285 6350 6493 6648 3825 2370 3286 2656 c 4170 c 4380 c 5003 c 154 753 1058 1087 1631 1815 2150 2350 2373 2444 2795 3633 3687 5609 5944 6197 75 276 288 329 412 493 598 1423 2837 2947 2990 3002 3942 4129 4880 5200 5573 5989 215 302 651 839 902 1279 1768 2502 2758 2770 3946 4252 4753 5141 5329 5482 5540 5871 6154 6270 6333 1878 2634 4619 662 747 1089 1324 1726 1879 2208 2635 2639 2675 3229 3997 4075 4156 4165 4243 4620 5119 5155 5172 5430 5476 5494 5835 5940 5952 6030 6038 6049 6124 6702 967 1124 1505 1625 2673 2809 c 3649 c 4187 4397 4477 c 5020 5129 5207 5962 6033 c 6185 c 6697 c 665 2392 2678 3735 5284 5433 6356 1659 2541 2360 11 65 1270 3908 703 c 870 c 1186 c 1613 c 1870 c 1929 c 1957 c 2029 c 2041 c 2065 c 2098 c 2165 2380 2577 c 2617 2657 c 2921 3261 c 3269 3285 c 3563 c 3569 c 3593 3599 3606 c 3609 c 3621 c 3741 c 3877 c 4234 c 4427 4776 c 4835 5429 c 5635 c 5782 5863 6263 6513 c 6587 11 65 1270 3908 161 784 830 849 917 1052 1067 1164 1230 1458 1472 1488 1587 1823 2130 2531 2592 2738 3009 3107 3124 3135 3147 3158 3681 4670 4851 5223 5588 5627 5862 5915 5929 5934 5986 519 4263 4545 4584 5031 5390 5549 1775 2023 2112 2174 3308 3932 4700 5166 610
4. Enzyme of cuts Positions c indicates the complementary strand Aatl Acc651 Accl Acil Acsl AcyI Alul Alw44AI AlwI AlwNI Aosl ApaLl Apol Asel Asp700 Asp718 AspEI AspHI Aspl 1 5 2 2 74 N CA A 33 18 N N C A 3631 279 332 415 601 4883 2366 3282 1548 2379 129 212 240 252 266 399 433 524 c 557 c 669 690 c 767 c 1945 c 1993 2005 c 2065 2110 2388 c 2392 2669 2730 c 2744 c 2747 c 2775 2802 3180 c 3206 c 3219 3227 c 3295 c 3480 3492 3501 3513 3523 3534 3580 3735 3798 3892 c 3956 c 4057 c 4060 c 4300 4340 c 4345 4395 c 4411 4437 4493 c 4552 4624 4662 4688 4698 4737 4911 c 4958 5057 c 5166 c 5243 5287 5408 c 5454 5645 c 5736 c 6098 6107 c 6242 6352 c 6473 c 6492 c 6619 c 6647 c 1175 1232 2354 2477 3131 3142 276 329 412 598 3826 4528 4880 5262 829 848 1051 1822 3680 1421 2360 728 759 834 1048 1775 1814 1818 1909 1929 2023 2174 2248 2282 2310 2320 2341 2516 2861 3118 3308 3596 3650 3932 4390 4751 4770 5449 5512 5612 6133 6390 6436 6526 4633 5130 6376 1721 c 1887 2634 c 2643 3237 4005 4070 c 4251 4615 c 4628 5163 5167 c 5484 5947 c 5948 6044 c 6046 6132 1778 1891 1912 6281 2697 3236 3928 5579 4633 5130 6376 1175 1232 2354 2477 3131 3142 161 5627 161 5627 5202 2366 3282 5802 730 2343 3939 4129 4637 5134 5219 6380 3944 1721 4508 25 8007 26UM Chapter 11 Rev A 2003 E
5. Qu GFP Assays 25 8007 26 25 8007 49 25 8010 36 25 8010 37 Aessy Hd 931d d43 i Amersham e KY Biosciences Bio _ Image um 25 8007 26UM Rev A 2003 fw GFP Assays 25 8007 26 25 8007 49 25 8010 36 25 8010 37 am 25 8007 26UM Rev A 2003 GFP PLCG PH domain Assay Amersham I e Biosciences Bio Image GFP PLC8 PH domain Assay 25 8007 26 25 8007 49 25 8010 36 25 8010 37 GFP PLCG PH domain Assay Amersham Y Biosciences um 25 8007 26UM Rev A 2003 Front cover Top image Basal distribution of GFP PLC6 PH domain CHO derived cell line stably expressing GFP PLC8 PH domain imaged before the addition of agonist The GFP fusion protein is most concentrated at the plasma membrane DRAQ5 nuclear stain also shown Bottom image Agonist induced distribution of GFP PLC PH domain CHO derived cell line stably expressing GFP PLC8 PH domain imaged 20 s after the addition of 300 uM ATP The amount of GFP fusion protein at the plasma membrane is decreased after treatment DRAQ5 nuclear stain also shown 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 mm vr 25 8007 26UM Page finder Rev A 2003 Page finder Chapter 1 Introduction 1 1 Hipnl ISl
6. 9 38 50 34 9 38 50 34 851 4998 8 51 49 88 922 47 06 9 22 47 06 8 756 4554 8 76 46 64 x 84 46 31 84 4631 5 8 53 44 23 8 63 44 23 6 09 46 6 09 4B 115 4557 115 45 57 1D 23 43 7 1 23 437 g E stimulated 1248 46 96 12 48 46 96 v m J unstimulated stimulated E 3d mean 8 88 45 38 sd 1 83 2 62 92 z 0 64 24 24 A M lt IPN 621002p unstim rds O21002pic stimrdS results 1 Isl Fig 5 4 Exported data and manipulation in Microsoft Excel Table 5 1 Results from a typical single assay performed using the suggested protocol Signal to noise is mean signal mean background background standard deviation ref 28 Magnitude of response is mean signal mean background CV is standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of assay quality ref 28 n tu um 25 8007 26UM Chapter 5 Rev A 2003 5 4 Assay characterization 5 4 1 Translocation index Translocation data presented throughout this manual are expressed in terms of the Translocation Index which reports the agonist induced translocation of GFP PLC PH domain fusion protein from the cell membrane to the cytoplasm The translocation index is obtained by taking the negative of the Dpeak reported in the population data file of the Plasma Membrane Trafficking analysis module Translocation index Dpeak 5 4 2 Summary of quanti
7. added to required wells After 60 min incubation the microplates are placed into the IN Cell Analyzer 3000 The Plasma Membrane Trafficking analysis module is used to image each well A baseline image is taken followed by a series of sequential images taken every 5 s for 30 s after the addition of the reference agonist to each well This provides a timecourse of response for each well Alternatively a baseline image can be taken followed by addition of the reference agonist followed by a final image approximately 20 25 s after addition of agonist time for maximum response START Seed cells Leave at room temp for 1 h optional Incubate overnight 37 5 v Decant Wash Decant Add Assay medium with nuclear stain Add test compounds and controls Incubate 60 min 37 C 5 CO Image plates on addition of ATP on the IN Cell Analyzer 3000 using Plasma Membrane Trafficking analysis module maximum response time 25 s Remove from IN Cell Analyzer 3000 25 8007 26UM Chapter 5 Rev A 2003 5 2 4 Detailed antagonist assay protocol 96 well format NOTE whenever possible keep the microplate at 37 5 and 9596 humidity 1 The day before starting the assay seed 2 0 x 10 cells per well in 200 ul per well of Growth medium Incubate at room temperature for 1 h optional before incubating for 24 h at 37 5 If one of the wells on the cell plate is used for flat field
8. 4 replicates per data point Fig 5 6 ATP dose response curve using the supplied GFP PLC PH domain cell line The calculated ECzo was 5 2 uM Hill Slope 0 95 Error SD n 4 replicates per data point 25 8007 26UM Chapter 5 Rev A 2003 Parameter Assay Data SD Assays Replicates Signal to Noise 16 62 x 5 13 14 24 Z factor 0 40 0 17 14 24 Magnitude of Response 26 59 6 68 14 24 CV Stimulated 8 69 1 91 14 24 Unstimulated 14 47 3 14 14 24 5 4 3 Seeding density Fig 5 5 shows the effect of varying seeding density in a 96 well microplate The data were collected 25 s after the addition of 300 uM ATP Significant differences between stimulated and non stimulated cells were seen at cell densities ranging from 1 5 x 104 to 2 5 x 10 cells per well For the greatest assay signal we recommend seeding in the range of 1 5 x 10 to 2 0 x 104 cells per well E Unstimulated 604 ig Stimulated Translocation index 15 000 20 000 25 000 5 4 4 ATP dose response Fig 5 6 shows an agonist dose response curve for the supplied cells to ATP The data were collected from Frame 0 and Frame 6 25 s after addition of agonist and demonstrate an of 5 2 uM 40 gt lt B o2 2 e 1 E S 10 04 an 7 5 4 3 ATP conc M Fig 5 7 Time course of GFP PLC PH domain translocation using ATP as an agonist Maximal respon
9. Biolmage A S under patents US 6 172 188 EP 851874 and EP 0896753 and other pending and foreign patent applications and under license from Vertex Pharmaceuticals formerly Aurora Biosciences Corporation under patents US 5 625 048 US 5 777 079 US 5 804 387 US 5 968 738 US 5 994 077 US 6 054 321 US 6 066 476 US 6 077 707 US 6 090 919 US 6 124 128 US 6 172 188 European patent 1104769 and Japanese patent JP3283523 and other pending and foreign patent applications The CMV promoter is covered under US patents 5 168 062 and 5 385 839 and its use is permitted for research purposes only Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation 214 Technology Innovation Center Iowa City IA52242 USA This product is sold under license from Columbia University under US patent Nos 5 491 084 and um 25 8007 26UM Chapter 2 Rev A 2003 6 146 826 Rights to use this product as configured are limited to internal use for screening development and discovery of therapeutic products NOT FOR DIAGNOSTIC OR THERAPEUTIC USE IN HUMANS OR ANIMALS No other rights are conveyed For customers wishing to use the assay for screening for potential therapeutic agents attention is drawn to the existence of US Patent Number 6 054 280 Methods for Diagnosis and Treatment of PH Domain Signal Transduction Disorders issued 25 April 2000 and assigned to Sugen Inc CA USA All goods and services are sold subj
10. 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 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 suitably equipped laboratory environment and should only be used 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 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 25 8007 26UM Chapter 4 Rev A 2003
11. ml The vector should have the characteristics outlined in Table 7 2 Property Value Limits Measurement method Concentration 250 pg ml UV Absorbance 260 nm in water Purity Minimal Ax60 A2809 ratio Between UV Vis contamination of the 1 8 2 2 Absorbance DNA construct by RNA 260 nm and or protein 280 nm Expected restriction The restriction Agarose gel pattern digests should give electrophoresis fragments of the sizes shown in Table 7 3 Enzyme s of cuts Fragment s size bp Puz1 3 1938 2013 2755 Nbe1 1 6706 Nco1 6 296 719 751 964 1014 2962 Not1 1 6706 1 2 944 5762 Prohlem Low assay response positive vs negative controls e Low nuclear intensity Image is out of focus o Cells do not adhere to well bottom in plate Shading across image field 25 8007 26UM Chapter 8 Rev A 2003 Chapter 8 Troubleshooting guide 8 1 Troubleshooting guide Possible causes and remedies 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 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
12. other than the IN Cell Analyzer 3000 25 8007 26UM Chapter 4 Rev A 2003 Chapter 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 CAUTION Contains genetically modified material Genetically modified cells supplied in this package are for use 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
13. with the assay 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 is added to the medium to maintain proper pH 1 5 Repeat assay with fresh reagents 1 6 Use actively growing cells maintained at 37 C Pre warm reagents to 37 Possible cause 2 1 Nuclear stain concentration too low 2 2 Nuclear stain incubation time too short Remedy 2 1 Adjust Nuclear stain concentration to recommended level 2 2 Adjust Nuclear stain incubation time to recommended length Possible cause 3 1 Autofocus Offset is chosen incorrectly or the system may need to be realigned Remedy 3 1 Alignment and calibration of instrument Perform Z stack on cells Change Autofocus Offset Possible cause 4 1 Seeding density too high Remedy 4 1 Reduce seeding density Possible cause 5 1 flat field correction not applied or flat field solution too weak Remedy 5 1 Apply flat field correction or adjust flat field solution 25 8007 26UM Chapter 9 Rev A 2003 Chapter 9 References 9 1 References 1 Homma Y et al Tissue and cell type specific expression of mRNAs for four types of inosit
14. 0 648 1118 c 1856 1975 c 2002 c 3326 c 3398 c 3462 c 3977 4509 2453 2546 c 730 1173 2064 2103 2343 2877 3772 3865 3939 4129 4191 4637 5134 5219 6380 25 8007 26UM Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand BspDI BspHI BspMI BspWI BsrBI BsrDI BsrFI BsrGI Bsrl BssHII Bst11071 BstBI BstNI BstUI BstXI BstYI Cfol Cfr10I Clal Csp451 Csp61 Ddel Dpnl Dpnll Dral Drall Dralll Drdl Dsal DsaV 2 3 4 39 N 20 19 13 29 13 10 31 31 2638 4607 4857 4962 5970 878 c 3713 c 4094 4544 137 244 366 398 437 530 554 803 1054 2059 2191 2200 2245 2677 2707 2739 2741 2783 2810 2840 3377 3449 3500 3579 3585 3817 3901 3924 4063 4069 4186 4222 4269 4536 4632 5684 6072 6644 6692 1945 2804 c 4439 c 4493 4960 c 66 c 4059 5568 5742 c 2843 4145 4326 5717 97 1375 449 c 887 940 1034 c 1129 1252 2192 2261 3037 3517 c 3770 3971 5157 5327 c 5596 5639 5757 6163 6275 c 6288 c 4223 1549 1721 4508 244 437 3346 3401 3418 4213 6531 6544 6665 214 2362 2720 2744 2764 3140 3227 3892 4193 4225 4626 4706 4809 4811 4911 5243 5736 6066 6647 4547 1726 1879 3229 3997 4243 4620 5155 5172 5940 5952 6038 6049 6702 1891 2142 2698 2722 2735 2744 2766 2792 2800 3237 3820 3828 3892 3929 4195 4225 4227 4455 4708 4811 4911 5243 5580 5673 6066 6175 6349 6449 6516 2843 4145 4326 5717 2638 4607 1721 4508 98 372 452 485 536 701 10
15. 007 26UM Chapter 1 Rev A 2003 Stimulated cell GFP PLC6 PH domain translocates transiently to cytoplasmic space 25 120 s Post stimulated cell GFP PLC PH domain re accumilates at plasma membrane 25 8007 26UM Chapter 2 Rev A 2003 Chapter 2 Licensing considerations 2 1 Product right to use Use of the GFP PLC6 PH domain Assay is limited as stated in the terms and conditions of sale These vary in accordance with the product code purchased Description Product code Non commercial educational scientific 25 8010 36 Research for the discovery and development of human therapeutics 25 8007 49 Screening for the discovery and development of human therapeutics 25 8007 26 Assay Evaluation for 6 month period 25 8010 36 for 12 month period 25 8010 37 2 2 Legal Cy is a trademark of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc 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 FACS is a trademark of Becton Dickinson and Co Oracle is a trademark of Oracle Corporation Cellomics and ArrayScan are trademarks of Cellomics Inc Hoechst is a trademark of Aventis Geneticin is a registered trademark of Life Technologies Inc DRAQS is a trademark of Biostatus Limited This product was developed with and sold under license from
16. 32 558 c 836 c 952 c 1068 c 1826 c 1996 2023 2384 3004 3012 c 4491 c 5811 6314 c 1424 1424 3378 3450 5200 5573 839 2325 3879 665 2678 5433 1775 2023 2174 3308 3932 4857 4962 5970 99 373 453 486 537 702 1064 1377 2368 3284 4132 4645 5321 4342 730 2343 2378 4170 c 4380 c 662 747 1089 1324 1726 1879 2208 2635 2639 2675 3229 3997 4075 4156 4165 4243 4620 5119 5155 5172 5430 5476 5494 5835 5940 5952 6030 6038 6049 6124 6702 237 430 1670 1753 1837 2007 2107 2233 2666 2954 4342 4822 5438 5660 5677 5756 1064 5321 244 437 1841 2012 2384 2385 2644 3346 3401 3418 3830 3990 4213 4730 4765 5266 5617 6313 6531 6544 6665 3399 511 c 1195 c 2167 c 2475 c 3167 c 3207 3389 3461 3784 c 4039 c 4125 4189 4255 c 4464 25 8007 26UM Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand SfaNI Sfcl Sfil Sful Smal SnaBI Snol Spel Sphl SspBI Sspl Stul Styl Tagl Thal Tru9I Tsp509I Tth1111 Van9 11 Xbal Xholl Xmal Xmalll Xmnl 25 8007 26UM 20 11 1 N 20 19 35 22 4648 c 4742 5101 c 5350 5541 c 6593 c 835 1080 1865 1883 2249 2321 2725 3875 5556 6234 6425 3585 1721 4508 2385 494 4633 5130 6376 153 3380 3452 4231 97 1375 6 53 3156 4997 3631 514 1265 2229 3243 3539 3632 4258 824 945 1467 1721 2034 2379 2397 2638 2913 3675 3939 4095 4119 4155 4317 4508 4607 5148 6592 6697 1464 1578 2260 365
17. 4 su rerom mk 2 n Rm 544 5 TH 2 uus maman eoa mib ee ca 5 4 6 Sensitivity of assay to DMSO Ethanol and Methanol 5 4 7 Effects of different culture conditions 5 4 8 96 well microplate s abilllV lt Chapter 6 Vector use details 6 1 General guidelines for vector use 6 2 Transient transfection with pCORON1000 GFP PLC PH domain 6 3 Stable cell line generation with pCORON1000 GFP PLC8 PH d ma prere trenon ie ueu a E E EE Chapter 7 Quality Control 7 1 GFP PLC PH domain cell line 1 2 GFP PL08 PH domain expression vector Chapter 8 Troubleshooting Guide 8 1 Troubleshooting guide Chapter 9 References 9 1 REPERANGES REED LUE ra de Chapter 10 Related Products 10 1 Related productis 25 8007 26UM Page finder Rev A 2003 Chapter 1 1 Appendix 11 1 Appendix A Restriction map of pCORON1000 GFP PLC8 PH domain ac etree sede ee sane 25 8007 26UM Chapter 1 Rev A 2003 Chapter 1 Introduction 1 1 Introduction In mammals the delta isoform of phospholipase C PLCS is expressed in a variety of tissues 1 It has been shown that genes homologous to that encoding mammal
18. 4 4311 4445 4604 214 2362 2720 2744 2764 3140 3227 3892 4193 4225 4626 4706 4809 4811 4911 5243 5736 6066 6647 161 784 830 849 917 1052 1067 1164 1230 1458 1472 1488 1587 1823 2130 2531 2592 2738 3009 3107 3124 3135 3147 3158 3681 4670 4851 5223 5588 5627 5862 5915 5929 5934 5986 172 786 1040 1137 1148 1175 1232 1517 1596 1659 2354 2477 2541 3131 3142 3168 3386 3458 3550 5369 5624 5930 3944 1932 2372 1726 1879 3229 3997 4243 4620 5155 5172 5940 5952 6038 6049 6702 2383 2389 3732 5202 Chapter 11 Rev A 2003
19. 48 214 2362 2720 2744 2764 3140 3227 3892 4193 4225 4626 4706 4809 4811 4911 5243 5736 6066 6647 984 c 1811 1840 1883 2198 c 3483 c 4150 4175 4720 c 5363 5650 5831 2697 3236 3928 5579 1892 2793 2801 3829 6450 11 65 238 431 1270 1671 1839 1863 2391 2667 2956 3098 3248 3573 3579 3588 3631 3734 3908 4299 4326 4551 4824 5411 5678 5758 6216 6650 6668 6679 688 2228 c 2726 4536 4712 5270 6000 c 6578 c 730 2343 3939 4129 4637 5134 5219 6380 1891 2142 2698 2722 2735 2744 2766 2792 2800 3237 3820 3828 3892 3929 4195 4225 4227 4455 4708 4811 4911 5243 5580 5673 6066 6175 6349 6449 6516 1889 2140 2696 2720 2733 2742 2764 2790 2798 3235 3818 3826 3890 3927 4193 4223 4225 4453 4706 4809 4909 5241 5578 5671 6064 6173 6347 6447 6514 678 1588 2380 2532 678 1588 2380 2532 757 3648 564 842 958 1074 1464 1578 1832 1988 2015 2260 2376 2996 3018 3654 4311 4445 4497 4604 5803 6320 1588 2532 1840 2012 2384 2643 2844 3731 3808 3830 3858 3989 4079 4146 4327 4730 4764 5265 5507 5617 5684 5718 6122 6312 6338 6485 530 1169 1208 1214 1457 1930 2948 4004 c 4782 c 4791 c 5075 c 5110 5316 c 5732 5959 25 8007 26UM Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand Ital Kasl Kpzl Ksp6321 Mael Maell Maeln Maml Mbol Mboll McrI Mfel Mlul MIuNI Mscl Msel Msll MspA1l Mspl Munl Mual Mwol Nael Narl Ncil 25 8007 26UM 44 16 18 21 31
20. 63 1376 2367 3283 4131 4644 5320 2221 3290 3592 4489 4640 4875 5301 5841 6007 6416 664 749 1091 1326 1728 1881 2210 2637 2641 2677 3231 3999 4077 4158 4167 4245 4622 5121 5157 5174 5432 5478 5496 5837 5942 5954 6032 6040 6051 6126 6704 662 747 1089 1324 1726 1879 2208 2635 2639 2675 3229 3997 4075 4156 4165 4243 4620 5119 5155 5172 5430 5476 5494 5835 5940 5952 6030 6038 6049 6124 6702 1489 2131 2593 5224 5916 5935 4822 2191 2951 818 1751 2014 2995 3669 3853 4719 6588 514 1265 2229 3243 3539 4258 242 435 1839 2010 2382 2383 2642 3344 3399 3416 3828 3988 4211 4728 4763 5264 5615 6311 25 8007 26UM Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand DsaV 21 Eael 10 Eagl 2 11051 1 Earl 4 Ecl1361I 2 EclX1 2 EcoS7I 8 109 1 EcoRI 1 EcoRII 9 Esp3l 2 Fnu4HI 44 FnuDII 19 FokI 12 FspI 4 Haell 5 Hael 30 Hgal 8 HgiAl 8 Hhal 29 HinP1I 29 Hincll 4 Hindll 4 HindIII 2 Hinfl 20 Hpal 2 Hpall 24 Hpbhl 15 6529 6542 6663 9 63 1268 2389 3732 3906 4297 4324 4549 5409 2389 3732 5802 2656 c 4170 c 4380 c 5003 c 728 2341 2389 3732 1449 1916 1934 2354 3972 4404 5136 6148 c 4822 2354 242 435 3344 3399 3416 4211 6529 6542 6663 4765 4807 c 835 1776 1892 1903 2113 2201 2321 2389 2392 2517 2699 2731 2745 2767 3238 3580 3735 3787 3798 3888 3893 3930 3971 4058 4061 4064 4300 4396 4437 4451 4552 4662 4771 5058 5287 5382 5409 5748 6076 6282 6285 6350 6493 66
21. 7 6352 1840 2012 2384 2643 2844 3731 3808 3830 3858 3989 4079 4146 4327 4730 4764 5265 5507 5617 5684 5718 6122 6312 6338 6485 1659 2541 244 437 3346 3401 3418 4213 6531 6544 6665 214 2362 2720 2744 2764 3140 3227 3892 4193 4225 4626 4706 4809 4811 4911 5243 5736 6066 6647 137 244 366 398 437 530 554 803 1054 2059 2191 2200 2245 2677 2707 2739 2741 2783 2810 2840 3377 3449 3500 3579 3585 3817 3901 3924 4063 4069 4186 4222 4269 4536 4632 5684 6072 6644 6692 2845 4328 3826 1841 2012 2384 2385 2644 3830 3990 4730 4765 5266 5617 6313 Chapter 11 Rev A 2003 Enzyme of cuts Positions c indicates the complementary strand Neol NdeI Ndell NgoMI Nhel NiaIV Noi Nsil Nspl NspV PfIMI Plel 1 Pmll Ppz101 Psp1406I PstI Pvul Pvull Real Rsal 1 Sapl Sau3AI Sau96I Scal ScrFI SexAI SfaNI 6 2 31 2 1 34 22 21 1 20 514 1265 2229 3243 3539 4258 388 1329 662 747 1089 1324 1726 1879 2208 2635 2639 2675 3229 3997 4075 4156 4165 4243 4620 5119 5155 5172 5430 5476 5494 5835 5940 5952 6030 6038 6049 6124 6702 2843 4326 1814 118 136 458 518 1102 1269 1343 1363 1558 1753 1793 1798 1935 2005 2233 2409 3247 3380 3452 3543 3700 4045 4231 4262 4288 4777 4861 4966 5359 5395 5473 5483 5974 6694 621 979 2009 2063 2227 2368 2876 2888 2909 3284 3350 3422 3827 3862 4622 4915 5505 5716 5757 5851 6623 6662 2389 3382 3454 3380 3452 4231 4777 1721 4508 19
22. 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 and users of these products MUST make themselves aware of and observe the Local Regulations or Codes of Practice which relate to such matters For further information refer to the material safety data sheet s and or safety statement s 4 2 Storage The pCORON GFP PLC PH domain expression vector NIF1991 should be stored at 15 C to 30 The CHO derived cells expressing the GFP PLC8 PH domain fusion protein NIF1954 should be stored at 196 C in liquid Nitrogen 4 3 Handling 4 3 1 Vector After thawing the DNA sample centrifuge briefly to recover the contents 4 3 2 Cells Care should be taken to ensure that the cells are not warmed if they are not being used immediately Do not centrifuge the cell samples upon thawing 25 8007 26UM Chapter 5 Rev A 2003 Chapter 5 Cell assay design 5 1 Culture and maintenance of CHO derived GFP PLC5 PH domain 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 Nutrient Mixture F 12 Ham medium with Glutamax Invitrog
23. T Sch rfe System GmbH is recommended to ensure accurate cell counting prior to seeding Alternatively a hemocytometer may be used Environmentally controlled incubator 5 CO 95 relative humidity 37 C Imager microscope e g IN Cell Analyzer 3000 Laminar flow cell culture bench Tissue culture flasks T flasks and pipettes Controlled freezing rate device providing a controlled freezing rate of 1 C per min Standard tissue culture reagents and facilities see also section 5 1 1 3 5 Instrument requirements The GFP PLC PH domain assay has been developed and optimized for analysis using the IN Cell Analyzer 3000 in conjunction with the Plasma Membrane Trafficking analysis module Please refer to the instrument user manual for details on instrument set up and the module manual for details on the algorithm settings For further information on either of these products please contact Amersham Biosciences um 25 8007 26UM e Chapter 3 Rev A 2003 25 8007 26UM Chapter 3 Rev A 2003 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 rapidly and at high resolution enabling high throughput and high content testing of drug compounds 3 5 2 Analysis of GFP PLCS PH domain assay on epifluorescence microscopes For speed
24. ate the medium from the cells 4 Gently resuspend the cells in Freeze medium until no clumps remain 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 h 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 13 2 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 16 6 hours under standard conditions Fig 5 1 15 In cell number o 0 25 50 75 100 125 Time hours 5 2 Assay set up 5 2 1 Live cell GFP PLCS PH domain assay using the IN Cell Analyzer 3000 This manual provides a suggested protocol to use the GFP PLC PH domain assay for antagonist screening on the IN Cell Analyzer 3000 5 2 2 Microplate set up for 96 well format assays The GFP PLC PH domain assay is optimized for use in an antagonist format It is recommended that actively growing cells are used which are maintained at 37 for the duration of the assay Reagents used during the assay should be pre warmed to 37 C It is essential that the number of cells Fig 5 2 Flow diagram showing a hasic protocol suitable for a GFP PLC8 PH domain anta
25. correction it should not contain cells 2 On the day of the assay prepare the Wash medium and Assay medium Prepare a 4 fold concentrated stock solution of the reference agonist compound ATP at 1200 uM in Assay medium 3 Prepare 3 fold concentrated stock solutions of test compounds in Assay medium Solvent vehicle controls are also prepared in Assay medium if required 4 Decant the overnight Growth medium from the cells and add 200 ul of Wash medium to each well Decant the Wash medium and add 100 ul of Assay medium into each well 5 Add 50 ul of the prepared 3 fold concentrated stocks of the test and control compounds to the appropriate wells The total well volume is 150 ul 6 Incubate the microplates at 37 C 5 for 60 min 7 Read the assay microplate using the IN Cell Analyzer 3000 and the Plasma Membrane Trafficking analysis module Perform a series of 7 sequential reads Toto T for each well imaging every 5 s Dispense 50 ul of 4 fold concentrated reference agonist 1200 uM ATP to each well immediately after the To read Alternatively a baseline image can be taken followed by addition of the reference agonist followed by a final image approximately 20 25 s after addition of agonist time for maximum response The total well volume is 200 ul 8 Perform the data analysis using the IN Cell Analyzer 3000 Plasma Membrane Trafficking analysis module 5 2 5 Timing schedule When performing a screen rather
26. culating Z factor Assay performance can be assessed by calculating the Z factor a dimensionless value defined by Zhang et al 28 Using the IN Cell Analyzer 3000 a Z factor of 0 3 should be obtained with the assay under standard conditions if the experiment is performed as described in this manual 430 Mc Ue Zi 1 where o standard deviation u mean signal ct positive control c negative control 5 3 2 Example results The following figures are taken from a set of experiments to give the user an overall view of the images and results that can be obtained with the GFP PLC PH domain assay Fig 5 3 shows images taken on the IN Cell Analyzer 3000 of the supplied CHO derived GFP PLC8 PH domain expressing cells before and after stimulation with 300 uM ATP Following image analysis the population data is exported into Microsoft Excel for further manipulation Fig 5 4 SS RE mms sms A RAV POPULATION DATA TRANSLOCATION INDEX Dpeak 2 junstimulated stimulated unstimulated stimulated Dpeak 96Dpeak Dpeak 998 4873 9 98 48 73 8 91 44 9 8 31 44 3 452 42 93 4 52 42 93 1023 4711 11 23 4741 87B 48 35 8 76 48 35 5 93 4202 5 99 4202 9 53 44 14 9 53 44 14 888 43 48 9 98 43 48 7 34 44 88 734 44 89 12 06 45 2 12 06 45 2 872 r ze xe E Translocation of GFP PLC amp PH domain from ES TAT 4833 Ti 18 33 membrane to cytoplasm 86 AAT 86 AAT
27. des a specific probe for activation of PIP2 metabolism 16 17 PLC81 hydrolyses PIP2 generating two second messengers inositol 1 4 5 triphosphate IP3 and diaglycerol DAG The PLC 1 PH domain facilitates processive hydrolysis of PIP2 by tethering the catalytic core of this enzyme to the membrane surface 16 The IP3 released into the cytoplasm mobilizes Calcium from internal stores and promotes an influx of external Calcium whereas DAG activates protein kinase C 1 12 18 19 In resting cells PLC81 is localized at the plasma membrane similar to other PLC isoforms PLC81 activity is feedback inhibited by IP3 which has an eight fold higher affinity for PLC81 than does PIP2 20 This manual describes a live cell screening assay for agonist induced activation of PLC PH using a probe that incorporates the proprietary GFP green fluorescent protein variant The assay employs Redistribution technology to monitor the intracellular translocation of a GFP PLC PH domain fusion protein in a stably transfected mammalian cell line GFP PLC PH is associated with the inner surface of the plasma membrane in resting cells and translocates transiently into the cytoplasmic space upon pathway stimulation by an agonist Further general pathway information follows in the biology schematics below Fig 1 1 and Fig 1 2 This assay has been optimized to be read on the IN Cell Analyzer 3000 system using the Plasma Membrane Trafficking acqui
28. domain 29 pCORON1000 GFP PLC PH domain has been used to generate stably transfected cell populations The magnitude of 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 Table 7 1 Quality control information for GFP PLC PH domain cell line Table 7 2 Quality control information for the GFP PLC PH domain expression vector Table 7 3 Expected restriction pattern for the GFP PLC PH domain expression vector um 25 8007 26UM Chapter 7 Rev A 2003 Chapter 7 Quality control 7 1 GFP PLCS PH domain cell line The GFP PLC PH domain 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 should have the characteristics detailed in Table 7 1 Property Value Measurement method Assay stability Magnitude of response Quality Control Assay gt 20 for 20 passages after dispatch Z factor z 0 3 Viability from frozen gt 80 CASY1 Cell Counter and Analyzer System Model TT Cell diameter mm 12 15 CASY1 Cell Counter and Analyzer System Model TT Fluorescence at gt 30 000 for 20 FARCyte Gain 62 9 x 104 cells per ml passages after RFU dispatch 1 2 GFP PLC PH domain expression vector The GFP PLC PH domain expression vector is supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 0 at 250 pg
29. e dihydrofolate reductase DHFR gene that allows selection of expressing cells with methotrexate MTX 3 2 2 CHO derived GFP PLCS PH domain expressing cell line CHO derived cells were transfected with the pCORON1000 GFP PLC PH domain vector supplied using a lipofectamine based method A stable clone expressing the recombinant fusion protein was selected using 500 ug ml Geneticin The isolated clone was grown for 30 passages before being sorted using a FACS machine The cells were grown for a further 3 passages before freezing The cells tested negative for mycoplasma bacteria and yeast contamination testing details are available upon request The hIR expression appears to be extremely stable in CHO cells without MTX selection pressure the cell line having retained Insulin sensitivity for several passages in the absence of MTX MTX selection pressure is not recommended with this particular cell line because CHO cells may develop MTX resistance by several reported mechanisms including alterations to DHFR and decreased membrane transport 25 If the ability to exert continued selection pressure is desired then a DHFR deficient CHO cell is recommended along with a selection medium lacking Glycine Hypoxanthine and Thymidine 26 The present hIR expression levels in these cells are unknown However the cell line is responsive to both Insulin and Insulin like growth factor 1 IGF 1 in the low nanomolar range consistent with literature rep
30. e 96 well microplate one time series per well Translocation index um 25 8007 26UM Chapter 6 Rev A 2003 Chapter 6 Vector use details The plasmid vector pCORON1000 GFP PLC PH domain Fig 3 1 can be used to transiently or stably express the GEP PLC PH domain fusion protein in the cell line of choice 6 1 General guidelines for vector use pCORON1000 GFP PLC PH domain has been used successfully to express GFP PLC8 PH domain fusion protein both transiently and stably in the CHO 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 pCORON1000 GFP PLC5 PH domain 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 GFP PLC PH domain into CHO hIR For more information refer to manufacturer s guidelines for the desired transfection reagent 6 3 Stable cell line generation with pCORON1000 GFP PLC5 PH domain 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
31. ect to terms and conditions of sale of the company within the Amersham Biosciences group which supplies them A copy of these terms and conditions is available on request Amersham Biosciences UK Limited 2003 All rights reserved http www amershambiosciences com Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 JNA UK Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany 25 8007 26UM Chapter 3 Rev A 2003 Chapter 3 Product contents 3 1 Components summary e CHO derived cells expressing the GFP PLC8 PH domain fusion protein 2 vials each containing 1 ml and 1 x 106 cells NIF1954 pCORON1000 GFP PLC PH domain expression vector 1 vial containing 10 ug DNA at 250 ug ml supplied in TE buffer 10 mM Tris 1 mM EDTA pH 8 0 NIF1991 User manual 3 2 CHO derived cell line expressing GFP PLCS PH domain fusion protein NIF1954 3 2 1 CHO derived parental cell line The cell line is of Chinese hamster ovary origin derived from CHO K1 ATCC CCL 61 cells 21 22 23 that have been stably transfected with a non mutated full length human Insulin receptor hIR 24 The cells were transfected by a non viral method and the hIR expression is under control of the metallothionein promoter The vector contains th
32. en life technologies 31765 027 or equivalent GIBCO Fetal Bovine Serum FBS Invitrogen life technologies 10099 141 or equivalent Heat inactivate serum by incubation in a water bath at 56 C for 30 min GIBCO Penicillin Streptomycin P S 5000 units ml penicillin G Sodium and 5000 ug ml Streptomycin Sulfate Invitrogen life technologies 15070 063 or equivalent Geneticin G418 Sigma G 7034 or equivalent GIBCO Trypsin EDTA in HBSS w o Calcium or Magnesium Invitrogen life technologies 25300 054 or equivalent 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 Bovine Serum Albumin BSA Sigma A 4503 or similar e 4 2 Hydroxyethyl piperazine 1 ethanesulfonic acid HEPES Sigma H 3375 or equivalent Adenosine 5 triphosphate Magnesium salt ATP Disodium salt Sigma A 9187 or similar e Hoechst 33342 Trihydrochloride Fluoropure grade Molecular Probes H 21492 DRAQS Biostatus Cy5 monocarboxyl dye Amersham Biosciences PA05111 Fluorescein Isothiocyanate FITC Molecular Probes F 1300 Coumarin Molecular Probes D 126 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 b
33. er 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 2 0 x 10 cells per well in a volume of 200 ul adjust the suspension to 10 x 10 cells per ml We recommend a seeding density of 1 5 2 0 x 104 cells per well for these assays 9 Dispense 200 ul of cells into each well of a 96 well microplate or 40 ul into each well of a 384 well plate except the well reserved for the flat field solution see IN Cell Analyzer 3000 users manual for further information 10 Optionally incubate the microplates undisturbed on a level surface for 1 h at room temperature approximately 20 This treatment may reduce edge effects 11 Incubate the plated cells for 24 h at 37 C before starting the assay NOTE If the cells are near confluence prior to trypsinization they should be Fig 5 1 Growth curve of CHO derived GFP PLC PH domain expressing cell line only points on the linear portion of the curve are shown Doubling time 16 6 h um 25 8007 26UM Chapter 5 Rev A 2003 split into two T flasks Actively growing cells will then be ready for seeding the following day 5 1 6 Cell freezing procedure 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 in section 5 1 5 3 Pellet the cells at approximately 300 g for min Aspir
34. gonist screen All incubations are performed at 37 unless otherwise stated 25 8007 26UM Chapter 5 Rev A 2003 per well in the assay plates is consistent in order to minimize assay variability ATP is used as a reference agonist with an ECso value of approximately 5 uM The GFP PLC PH domain assay can be used with either Hoechst or DRAQS as the nuclear stain All data in this manual has been generated using DRAQS as the nuclear marker Hoechst is a suitable alternative to DRAQ5 however sequential imaging is required on the IN Cell Analyzer 3000 due to the spectra overlap of GFP and Hoechst 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 microplate solely for this purpose Alternatively a designated well on each microplate can contain flat field solution When seeding the plate this well must not contain any cells if the auxiliary file flat field correction tool is to be applied in the analysis module 5 2 3 Schematic antagonist assay protocol for use with the IN Cell Analysis System Fig 5 2 shows a typical schematic of the antagonist assay The cells should be seeded in the appropriate microplate the day before the experiment Decant the medium and wash the cells decant wash medium and add assay medium to each well Test compound control buffer or solvent vehicle controls are
35. ian in yeast and plants play important roles in cell growth and or responses to environmental stress 2 3 One of the PLCS isoforms PLC81 is ubiquitously expressed in a variety of tissues 1 and has three lipid interacting domains a pleckstrin homology PH domain a C2 domain and a catalytic core domain 4 PLC 1 normally resides at the plasma membrane by virtue of its PH domain 5 6 7 8 The PH domain binds to phosphoinositides which are components of the cell membrane and inositol phosphates which are the head group of phosphoinositides 9 The PH domain of PLC81 PLC8 PH plays a critical role in its membrane targeting The subcellular distribution of PLC81 is under osmotic regulation in MDCK cells 8 and hypotonic shock will induce its dissociation from the plasma membrane 7 The hydrolysis of a minor membrane phospholipid phosphatidylinositol 4 5 biphosphate PIP2 by PLC 1 is one of the earliest key events in the regulation of various cell functions by more than 100 extracellular molecules 10 11 12 13 A major function of the PH domain in PLC 1 is to modulate enzyme activity and PIP2 has been identified as a ligand for the domain 14 PLC 1 also recognizes phosphatidylinositol phosphatidylinositol 4 phosphate PIP as well as PIP2 and carries out the Ca2 dependent hydrolysis of these inositol phospholipids 15 PLC 1 binds selectively to PIP2 over other phosphatidylinositol lipids and thus provi
36. ig 5 10 Reproducibility of GFP PLC PH domain translocation response across an entire 96 well microplate when imaging analyzing in kinetic mode Error SD 12 replicates per data point 25 8007 26UM Chapter 5 Rev A 2003 5 4 7 Effects of different culture conditions To determine the effects of varying culture conditions on the ATP induced GFP PLC PH domain fusion protein translocation the stable CHO cells were cultured for 7 d in either the recommended Growth medium or Dulbecco s modified eagle medium DMEM Invitrogen life technologies 32430 027 in either 10 5 or 1 v v FCS The cells were then assayed in the same media using the recommended procedure Note the final concentration of ATP was 30 uM The results shown in Fig 5 9 demonstrate that the assay is optimal in the recommended Growth medium 40 5 Unstimulated iz Stimulated gt lt 2g t O 204 Se 2 104 Ke 0 S amp lt c c A ae GU i SS s gy Growth media 5 4 8 96 well microplate stability The following experiment was performed to assess assay stability in 96 well format on the IN Cell Analyzer 3000 Cells were seeded into all wells of a 96 well microplate Each well of cells was assayed using the recommended procedure The final concentration of ATP was 300 uM The results shown in Fig 5 10 demonstrate that the assay response is stable throughout the imaging of an entir
37. nzyme of cuts Positions c indicates the complementary strand Aval Avall Avill BamHI Banl Banll BbrPI BbsI Bbul Bcgl Bfal Bfrl Bgll BglII Blnl 1 Bpml BpuAI BsaAI BsaBI BsaHI Bsal BsaJl BsaWI Bsgl BsiEI BsiHKAI BsiYI BsmAI BsmFI Bsml 12861 3 7 4 24 16 15 Q co wo 16 15 15 10 12 15 1834 2102 2383 1753 2007 2107 2233 4342 5438 5660 2697 3236 3928 5579 3632 4620 619 977 2061 2366 2907 3282 3825 3860 5849 730 2343 2877 4191 1424 962 821 c 1762 c 1878 c 1914 2099 c 2187 c 2307 c 2503 c 2710 2778 3249 3773 c 3899 3941 3957 c 4050 c 4462 4757 c 5368 c 5759 6062 c 6268 c 6271 c 6361 5264 c 154 753 1058 1087 1631 1815 2150 2350 2373 2444 2795 3633 3687 5609 5944 6197 829 848 1051 1822 3680 137 244 366 437 2707 3585 5684 6702 3632 730 1173 2064 2103 2343 2877 3772 3865 3939 4129 4191 4637 5134 5219 6380 1146 2099 5733 962 494 1424 2948 4130 1878 2634 4619 276 329 412 598 3826 4528 4880 5262 916 c 1737 c 5736 514 1265 2010 2103 2229 2383 3243 3344 3416 3539 3574 3583 3632 3989 4258 6530 3857 5506 6337 6484 1841 c 2186 c 665 2392 2678 3735 5284 5433 6356 730 2343 3939 4129 4637 5134 5219 6380 203 1932 2071 2187 2188 2729 3055 3540 3807 4351 4764 6212 6491 6657 6675 203 1932 2071 2187 2188 2729 3055 3540 3807 4351 4764 6212 6491 6657 6675 588 826 916 c 941 c 1737 c 3677 4765 4807 c 4960 c 5736 329 48
38. of screening and quality of the images obtained we recommend performing the GFP PLC8 PH domain assay on the IN Cell Analyzer 3000 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 6 Software requirements IN Cell Analysis System The Plasma Membrane Trafficking analysis module is used to measure the translocation of a fluorescent probe between the cellular plasma membrane and the cytoplasm The GFP PLC PH domain assay utilizes this algorithm to monitor the translocation of GFP labelled PLC8 PH domain from the plasma membrane to the cytoplasm in kinetic mode Analyzed data are exported as numerical files in ASCII format ASCII format data can be utilized by MicrosoftTM 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
39. ol phospholipid specific phospholipase C Biochem Biophys Res Commun 164 406 412 1989 2 Yoko o T et al The putative phosphoinositide specific phospholipase C gene PLC1 of the yeast Saccharomyces cerevisiae is important for cell growth Proc Natl Acad Sci USA 90 1804 1808 1993 3 Hirayama T et al gene encoding a phosphatidylinositol specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana Proc Natl Acad Sci USA 92 3903 3907 1995 4 Essen L O et al Crystal structure of a mammalian phosphoinositide specific phospholipase C delta Nature 380 595 602 1996 5 Paterson H F et al Phospholipase C delta 1 requires a pleckstrin homology domain for interaction with the plasma membrane Biochem J 312 661 666 1995 6 Yagisawa H et al Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C delta1 alter the ligand binding phospholipase activity and interaction with the plasma membrane J Biol Chem 273 417 424 1998 7 Yagisawa et al Phospholipase C delta and related molecules Biochem Soc Trans 27 652 657 1999 8 Fujii M et al Real time visualization of PH domain dependent translocation of phospholipase C delta1 in renal epithelial cells MDCK response to hypo osmotic stress Biochem Biophys Res Commun 254 284 289 1999 9 Yagisawa H et al Expression and characterization of an inositol 1 4 5 tri
40. orts 27 3 3 GFP PLCS PH domain expression vector NIF1991 The 6 7 kb plasmid pCORON1000 GFP PLC8 PH domain contains bacterial ampicillin resistance gene and a mammalian neomycin resistance gene Fig 3 1 The sequence of the construct is available on a CD upon request A detailed restriction map is shown in chapter 11 appendix A Bi Fig 3 1 Vector map of the supplied GFP PLC PH domain expression vector ApaLI 6376 CMV enhancer Ncol 514 promoter Hindlll 757 PstI 839 Ampicillin resistance gene Chimeric intron Ncol 1265 GFP PLC6 PH domain ApaLl 5130 ApaLl 4633 pCORON1000 GFP PLC5 PH domain BamHI 4620 6706 bp Aval 1834 Clal 4607 _ N Aval 2102 Synthetic polyA Ncol 4258 Nocol 2229 Neomycin resistance gene W NN PstI 2325 3879 SEcoRI 2354 um YAN HindIII 3648 mal 2383 Aval 2383 Ncol 3539 VI SV40 enhancer early promoter flor Smal 2385 SV40 late polyA Ncol 3243 Ct 538 3 4 Materials and equipment required The following materials and equipment are required but not provided Microplates For analysis using the IN Cell Analysis System Packard Black 96 Well ViewPlates Packard Cat 6005182 are recommended For assays in 384 well format please email incellanalyzer uk amershambiosciences com for recommendations A CASY 1 Cell Counter and Analyzer System Model T
41. r I receptor reside in different regions of a common binding site Proc Natl Acad Sci USA 15 4404 4408 1991 28 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 29 Freshney R I Cloning and Selection of Specific Cell Types in Culture of Animal Cells 3rd Edition Wiley Liss Inc Chapter 11 161 178 1994 25 8007 26UM e Chapter 9 Rev A 2003 um 25 8007 26UM Chapter 10 Rev A 2003 Chapter 10 Related products 10 1 Related products Product Name GFP Assays GFP MAPKAP k2 Assay EGFP 2x FYVE Assay AKT 1 EGFP Assay EGFP SMAD2 Assay CypHer pCORON 1000 VSV G Expression Vector pCORON 1000 SP VSV G Expression Vector CypHerS Labelled Anti VSV G Antibody CypHer5 NHS Ester 1 mg pack CypHer5 NHS Ester 5 mg pack IN Cell Analysis System IN Cell Analyzer 3000 Plasma Membrane Trafficking Analysis Module Code 25 8008 82 25 8010 21 25 8010 17 25 8010 46 25 8008 51 25 8009 92 PA45407 PA15401 PA15405 25 8010 11 63 0048 95 Chapter 1 1 Appendix 11 1 Anpendix A Restriction map of pCORON1000 GFP PLC5 PH domain The following enzymes do not cut the vector AccIII Agel Apal Ascl Bcll 11021 BseAl BsiWI 1201 BspEl BstEIL Bsu361 CellI 47 EcoNI EcoRV Espl Kspl Mrol Nrul Pacl PaeR7I PinAI Pmel PpuMI Sacll Sbfl SgrAL Swal Xcml Xbol
42. rate 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 min 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 T2162 and gently resuspend the cells with a 10 ml pipette until all clumps have dispersed 7 Aspirate the cell suspension and dispense 1 ml cells into a new culture vessel 5 1 5 Cell seeding procedure The following procedure is optimized for cells grown in standard T 75 and 1 162 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 flasks and 4 ml for T 162 flasks ensuring that all cells are in contact with the solution Wait for 3 10 min 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 T2162 and gently resuspend the cells using a 10 ml pipette until all clumps have dispersed 7 Count the cells using either a CASY1 Cell Counter and Analyz
43. se 3 ul 10 uM FITC and 20 ul 10 uM CyS in 200 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 106 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 water bath and swirl gently for 1 2 min until the contents are thawed Do not thaw the cells for longer than 3 min 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 sub culturing procedure Incubation 5 95 humidity 37 The cells should be passaged at a ratio of 1 10 when they are 70 confluent 25 8007 26UM Chapter 5 Rev A 2003 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 Aspi
44. se is seen 25 s after stimulation with 300 uM ATP Error SD n 4 replicates per data point Fig 5 8 The effects of DMSO Ethanol or Methanol on the GFP PLCS PH domain assay Error SD n 8 replicates per data point 25 8007 26UM Chapter 5 Rev A 2003 5 4 5 Time course Time course analysis was performed to determine the optimal stimulation time for the antagonist screening protocol Fig 5 7 shows an example of time course data for cells stimulated with 300 uM ATP The results indicate that the dynamic range of the assay is maximal at the incubation time chosen for the screening assay 25 s m Unstimulated A Stimulated amp 30 E c 222 se S o 10 0 T T T T T T T T T T T 1 0 30 60 90 120 150 Time s 5 4 6 Sensitivity of assay to DMSO Ethanol and Methanol The GFP PLC PH domain assay was performed in the presence of DMSO lt 1 Ethanol lt 3 or Methanol lt 3 As can be seen in Fig 5 8 solvents at these concentrations cause no significant decrease in the ATP induced translocation compared to control y Stimulated 40 E Unstimulated 30 es so of e 1 S 10 E 0 v de de de de c c oge Ade c c c c3 s EK Solvent Fig 5 9 The effect of culture conditions on the membrane to cytoplasm translocation of GFP PLCS PH domain Error SD n 24 replicates per data point F
45. sition protocol and data analysis module When the pathway is stimulated the GFP PLC PH domain Fig 1 1 PLCS PH domain signaling pathway provided with permission from BioCarta www hiocarta com 25 8007 26UM Chapter 1 Rev A 2003 fusion protein translocates from the plasma membrane into the cytoplasm causing an intracellular redistribution of fluorescence intensity The Plasma Membrane Trafficking analysis module allows this response to be quantified relative to an ATP agonist control ATP has a typical of 5 uM in this assay and the dose response concentration range is 0 3 300 uM The response of the PLC8 PH domain assay is transient reaching a maximum level 20 25 s after stimulation with 300 uM ATP and decreasing to near resting levels within 2 min The assay protocol provided is formatted for antagonist screening and requires pre incubation of cells with test compounds While the cells are in the IN Cell Analyzer 3000 for each well a baseline image is captured followed by a timeseries of 7 images data points at 5 s intervals after the addition of the reference agonist ATP On line image analysis allows quantification of the time dependent redistribution response Extracellular Cytoplasm Fig 1 2 Agonist induced redistribution of GFP PLCS PH domain from the plasma membrane to the cytoplasm Agonist 20 25s Un stimulated cell GFP PLC6 PH domain is most concentrated at the plasma membrane 25 8
46. sphosphate binding domain of phosphatidylinositol specific phospholipase C delta 1 J Biol Chem 269 20179 20188 1994 10 Rhee S G and Choi K D Regulation of inositol phospholipid specific phospholipase C isozymes J Biol Chem 267 12393 12396 1992 11 Cockcroft S and Thomas G M Inositol lipid specific phospholipase C isoenzymes and their differential regulation by receptors Biochem J 288 1 14 1992 12 Berridge M J Inositol trisphosphate and calcium signalling Nature 361 315 325 1993 13 Noh D Y et al Phosphoinositide specific phospholipase C and mitogenic signaling Biochim Biophys Acta 1242 99 113 1995 14 Lomasney J W et al Phosphatidylinositol 4 5 bisphosphate binding to the pleckstrin homology domain of phospholipase C delta1 enhances enzyme activity J Biol Chem 271 25316 25326 1996 15 Rhee S G et al Studies of inositol phospholipid specific phospholipase C Science 244 546 550 1989 16 Garcia P et al The pleckstrin homology domain of phospholipase C delta 1 binds with high affinity to phosphatidylinositol 4 5 bisphosphate in bilayer membranes Biochemistry 34 16228 16234 1995 17 Ferguson M et al Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain Cell 83 1037 1046 1995 18 Berridge M J Cell signalling A tale of two messengers Nature 365 388 389 1993 19 Hirose K et al Spa
47. tative assay parameters Summaries of typical assay data using 300 uM as the agonist are shown in Table 5 1 and Table 5 2 In particular Table 5 1 shows the results obtained from a single assay plate indicating the level of well to well variation Table 5 2 shows a summary of the results obtained from 14 assays performed by different operators on different occasions giving an indication of inter assay variation Parameter Assay Data Assays Replicates Signal to Noise 19 24 1 24 Z factor 0 46 1 24 Magnitude of Response 29 14 1 24 CV Stimulated 8 97 1 24 Unstimulated 12 06 1 24 c _ 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 mean signal mean background background standard deviation ref 28 Magnitude of response is mean signal mean background CV is standard deviation x 100 mean Z factor is a dimensionless characteristic useful for evaluation of assay quality ref 28 Fig 5 5 ATP induced GFP PLC5 PH domain translocation as a function of seeding density Stimulated cells were treated with 300 uM ATP Unstimulated cells were treated with buffer only Cells were imaged 25 s after ATP addition kinetic data from Frame and Frame 6 using the Plasma Membrane Trafficking analysis module Error SD n
48. than imaging a time series for each well the IN Cell Analyzer 3000 and the Plasma Membrane Trafficking analysis module can be used to group wells together and generate kinetic data from baseline and maximum response images only For each well a baseline image is taken followed by addition of the reference agonist followed by a final image approximately 20 25 s after addition of agonist time for maximum response By grouping a number of wells together kinetic data can be generated from each well of a 96 well microplate in 13 min data based on the use of DRAQS as nuclear stain and one tile per well For more information please refer to the IN Cell Analyzer 3000 user manual 5 2 6 Important considerations When performing an antagonist screen it is important to remember that the test compound added to the plates will be diluted by the agonist addition It is recommended that the cells incubate in the target test compound concentration prior to the addition of the agonist This means that the test Fig 5 3 CHO derived cells expressing GFP PLCS PH domain a before stimulation and b 20 s after stimulation with 300 uM ATP Only a fraction of the entire field of view is shown in each panel A 25 8007 26UM Chapter 5 Rev A 2003 compound concentration during the agonist induced translocation will be 75 of the target concentration Other options are available and can be determined by the user 5 3 Results 5 3 1 Cal
49. tiotemporal dynamics of inositol 1 4 5 trisphosphate that underlies complex Ca2 mobilization patterns Science 284 1527 30 1999 20 Lemmon M A and Ferguson K M Signal Dependent Membrane Targeting by Pleckstrin Homology PH Domains Biochem J 350 1 18 2000 21 Puck T T et al Genetics of somatic mammalian cells III Long term cultivation of euploid cells from human and animal subjects J Exp Med 108 945 956 1958 22 Kao F T and Puck T T Genetics of somatic mammalian cells IV Properties of Chinese hamster cell mutants with respect to the requirements for proline Genetics 55 513 524 1967 23 Kao E T and Puck T T Genetics of somatic mammalian cells VII Induction and isolation of nutritional mutants in Chinese hamster cells Proc Natl Acad Sci USA 60 1275 1281 1968 24 Hansen B F et al Sustained signalling from the insulin receptor after stimulation with insulin analogues exhibiting increased mitogenic potency Biochem J 315 271 279 1996 25 Flintoff W F et al Isolation and partial characterization of three methotrexate resistant phenotypes from Chinese hamster ovary cells Somatic Cell Genet 2 245 261 1976 26 Ryser H J and Shen W C Conjugation of methotrexate to poly L lysine as a potential way to overcome drug resistance Cancer 45 1207 1211 1980 27 Kjeldsen T et al The ligand specificities of the insulin receptor and the insulin like growth facto
50. ut not supplied Growth medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 v v FBS 1 v v Penicillin Streptomycin and 1 v v Geneticin working concentration 0 5 mg ml 25 8007 26UM Chapter 5 Rev A 2003 Freeze medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 v v FBS 1 v v Penicillin Streptomycin and 1096 v v DMSO Wash medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 mM HEPES 0 246 w v BSA Assay medium Nutrient Mixture F 12 Ham medium with Glutamax supplemented with 10 mM HEPES 0 2 w v BSA and Nuclear stain either 0 2 uM Hoechst or 1 uM DRAQS stock solution 100 mM ATP in deionized water dispensed into aliquots and stored at 15 C to 30 C For assays performed on the IN Cell Analysis System Flat field FF solution components Cy5 monocarboxyl dye PA05100 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS FITC 1 mM stock solution prepared in 10 v v DMSO 90 v v PBS Coumarin 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 flat field solution to give a fluorescent signal in each channel between 700 3300 counts For a Hoechst stained assay prepare an initial FF solution containing 3 ul 10 uM FITC and 20 ul 1 mM Coumarin in 200 ul PBS For a DRAQS stained assay u
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