Carboxyfluorescein FLICA Apoptosis Detection Kit Caspase Assay
Contents
1. 300 uL cell suspension Or if a larger cell volume was used add the 30X FLICA solution at a 1 30 ratio For example if 2 9 mL of cell suspension was used add 100 uL of the 30X FLICA solution forming a final volume of 3 mL Each investigator should adjust the amount of FLICA reagent used to accommodate their particular cell line and research conditions Mix the cells by slightly flicking the tubes Incubate cells for 1 hour at 37 C under 5 COs protecting the tubes from light As cells may settle on the bottom of the tubes gently resuspend them by swirling cells once or twice during this incubation time This will ensure an even distribution of the FLICA reagent among all cells If cells are to be monitored using Hoechst stain add 1 5 uL Hoechst stain 0 5 v v Incubate for 5 minutes at 37 C under 5 CO3 Add 2 mL of 1X wash buffer to each tube Gently mix Centrifuge the cells at lt 400 X g for 5 minutes at RT 1 800 829 3194 www immunochemistry com 15 24 25 26 27 28 Immunochemistry Technologies LLC FLICA Booklet Carefully remove and discard supernatants Gently vortex the pellets to disrupt any cell to cell clumping Resuspend cells in 1 mL 1X wash buffer Gently mix Centrifuge the cells at lt 400 X g for 5 minutes at RT Carefully remove and discard supernatants Gently vortex pellets to disrupt any cell to cell clumping Resuspend the cell pellets in 300 uL 1X wash2. Section 20 or 21 Hoechst stain is provided ready to use at 200 ug mL Warning Hoechst stain is a potential mutagen Use of gloves protective clothing and eyewear are strongly recommended When disposing flush sink with copious amounts of water see MSDS for further information 12 Fixative If the stained cell populations cannot be evaluated immediately upon completion of the FLICA staining protocol cells may be fixed and analyzed up to 24 hours later on a microscope or flow cytometer The fixative is a formaldehyde solution designed to cross link cell components and will not interfere with the carboxyfluorescein labeling once the FLICA reaction has taken place After labeling add the fixative into the cell solution at a 1 10 ratio For example add 100 uL fixative to 900 uL cells Section 20 21 or 23 Fixed cells may be stored on ice or at 4 C up to 24 hours Do not use ethanol based or methanol based fixatives to preserve the cells they will inactivate the FLICA label Never add the fixative until the staining and final wash steps have been completed Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 9 FLICA Booklet 13 Reconstitution of the 150X FLICA Stock The FLICA reagent is supplied as a highly concentrated lyophilized powder It must first be reconstituted in DMSO forming a 150X stock concentrate and then diluted 1 5 in PBS to form a final 30X working solution For best results t
3. store it covered at 2 8 C for up to 14 days If more buffer is needed please contact ICT at 1 800 829 3194 or 952 888 8788 for technical assistance or to order a replacement component Warning The wash buffer contains sodium azide which is harmful if swallowed or absorbed through the skin Sodium azide can react with lead and copper sink drains forming explosive compounds When disposing of Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 8 FLICA Booklet excess wash buffer flush sink with copious amounts of water see MSDS for further information 10 Propidium lodide Propidium iodide PI may be used to distinguish between live cells and dead cells either caspase negative or caspase positive PI stains necrotic dead and membrane compromised cells They may be viewed through a fluorescence microscope or analyzed on a flow cytometer Section 21 or 24 The dye excites at 488 492 nm and exhibits an emission maximum at 635 nm PI is provided ready to use at 250 ug mL Warning Propidium iodide is a potential mutagen Use of gloves protective clothing and eyewear are strongly recommended When disposing flush sink with copious amounts of water see MSDS for further information 11 Hoechst Stain Hoechst stain can be used to label the nuclei of dying cells after labeling with the FLICA reagent It is revealed under a microscope using a UV filter with excitation at 365 nm and emission at 480 nm
4. D Brady L C Dang N J Bump C R Ferenz S Franklin T Ghayur M C Hackett and L D Hammill 1994 Crystal Structure of the Cysteine Protease Interleukin 1 Converting Enzyme A p20 p10 2 Homodimer Cell 7178 343 352 Wilson K P J F Black J A Thomson E E Kim J P Griffith M A Navia M A Murcko S P Chambers R A Aldape S A Raybuck and D J Livingston 1994 Structure and mechanism of interleukin 1 beta converting enzyme Nature 370 270 275 Rotonda J D W Nicholson K M Fazil M Gallant Y Gareau M Labelle E P Peterson D M Rasper R Ruel J P Vaillancourt N A Thornberry and J W Becker 1996 The three dimensional structure of apopain CPP32 a key mediator of apoptosis Nature Struct Biol 3 7 619 625 Kumar S 1999 Mechanisms mediating caspase activation in cell death Cell Death and Differ 6 1060 1066 Thornberry N A T A Rano E P Peterson D M Rasper T Timkey M Garcia Calvo V M Houtszager P A Nordstrom S Roy J P Vaillancourt K T Chapman and D W Nicholson 1997 A combinatorial approach defines specificities of members of the caspase family and granzyme B Functional relationships established for key mediators of apoptosis J Biol Chem 272 29 17907 17911 Ekert P G J Silke and D L Vaux 1999 Caspase inhibitors Cell Death and Differ 6 1081 1086 Copyright 2002 Immunochemistry Technologies LLC FLICA Booklet www
5. PI induced and non induced 1 As discussed in Section 7 culture cells to a density optimal for apoptosis induction according to your specific induction protocol 2 Induce apoptosis following your protocol as mentioned in Section 8 Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 20 20 21 Immunochemistry Technologies LLC FLICA Booklet Culture an equal volume of non induced cells for negative control cell populations Make sure that all tubes of cells contain similar quantities of cells Ideally cells should be at 1 X 10 cells mL for FLICA labeling If necessary cells may be induced to undergo apoptosis at lower concentrations but then concentrated just prior to labeling to 0 5 2 X 10 cells mL by centrifugation for 5 minutes at lt 400 X g at RT Once induction is completed transfer 300 uL of each cell suspension to Sterile tubes Larger cell volumes can also be used as determined by each investigator however more of the FLICA reagent may be needed per sample Larger volume cell suspensions label nicely using 25 cm tissue culture flasks laid flat as the incubation vessel Add 10 uL 30X FLICA solution directly to the 300 uL cell suspension Or if a larger cell volume was used add the 30X FLICA solution at a 1 30 ratio For example if 2 9 mL of cell suspension was used add 100 uL of the 30X FLICA solution forming a final volume of 3 mL Each investigator should adjust the
6. amount of FLICA reagent used to accommodate their particular cell line and research conditions Mix the cells by slightly flicking the tubes Incubate cells for 1 hour at 37 C under 5 COs protecting the tubes from light As cells may settle on the bottom of the tubes gently resuspend them by swirling cells once or twice during this incubation time This will ensure an even distribution of the FLICA reagent among all cells Add 2 mL of 1X wash buffer to each tube Mix the cells Centrifuge cells at lt 400 X g for 5 minutes at room temperature RT Carefully remove and discard supernatant Gently vortex the cell pellet to disrupt any cell to cell clumping Resuspend the cell pellet in 1 mL 1X wash buffer Centrifuge cells at lt 400 X g for 5 minutes at RT Carefully remove and discard supernatant Gently vortex the cell pellet to disrupt any cell to cell clumping Resuspend the cell pellet in 400 uL 1X wash buffer Stain one 400 uL aliquot of FLICA treated induced cells with 2 uL PI Cells that are to be analyzed by the bicolor PI screening protocol cannot be fixed Set aside a second aliquot of FLICA treated induced cells that does not contain PI Mix cells 1 800 829 3194 www immunochemistry com 21 FLICA Booklet 22 Put samples on ice Keep bicolor cells at 2 C 8 C protected from light for up to 24 hours 23 For bicolor analysis measure fluorescein on the FL1 channel and red fluorescen
7. buffer higher volumes may be used if a larger staining cell volume was used Place cells on ice At this point the cells may be stained with propidium iodide PI for bicolor analysis Step 24 observed immediately Step 25 or fixed for future viewing Step 26 To exclude dead cells from the analysis 1 5 uL PI solution may be added at this point 0 5 v v Cells may then be viewed using a long pass filter with the excitation at 490 nm emission gt 520 nm PI has a maximum emission at 637 nm To view cells immediately place 1 drop of the cell suspension onto a microscope slide and cover with a coverslip go to Step 27 If not viewing immediately cells may be fixed for viewing up to 24 hours later If cell pellets were resuspended in 300 uL wash buffer add 30 uL fixative to each tube If cells were resuspended in a different volume add the fixative at a 1 10 ratio into the volume of cell suspension to be fixed For example if 3 mL was used add 300 uL fixative a Incubate cells for 15 minutes at RT in the dark b Dry cells onto a microscope slide c Briefly wash the cells with PBS d Cover cells with mounting media and coverslip e Store slides at 2 8 C up to 24 hours Go on to Step 27 Observe cells under a fluorescence microscope using a bandpass filter excitation 490 nm emission gt 520 nm to view green fluorescence Cells bearing active caspase enzymes covalently coupled to the FLICA reagent appear g
8. flat as the incubation vessel When ready to label with the 30X FLICA solution cells should be at least 5 X 10 cells 100 uL aliquot per microtiter plate well Density can be determined by counting cell populations on a hemocytometer Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 11 FLICA Booklet 5 Add 10 uL 30X FLICA solution directly to the 290 300 uL cell suspension 6 Or if a different cell volume was used add the 30X FLICA solution at a 1 30 ratio For example if 2 9 mL of cell suspension was used add 100 uL of the 30X FLICA solution forming a final volume of 3 mL Each investigator should adjust the amount of FLICA reagent used to accommodate their particular cell line and research conditions 7 Mix the cells by slightly flicking the tubes 8 Incubate cells for 1 hour at 37 C under 5 COs protecting the tubes from light As cells may settle on the bottom of the tubes gently resuspend them by swirling cells once or twice during this incubation time This will ensure an even distribution of the FLICA reagent among all cells 9 Add 2 mL of 1X wash buffer to each tube 10 Mix the cells 11 Centrifuge cells at lt 400 X g for 5 minutes at room temperature RT 12 Carefully remove and discard supernatant 13 Gently vortex the cell pellet to disrupt any cell to cell clumping 14 Resuspend the cell pellet in 1 mL 1X wash buffer 15 Centrifuge cells at lt 400 X g for 5
9. of the tubes gently resuspend them by swirling cells once or twice during this incubation time This will ensure an even distribution of the FLICA reagent among all cells 9 Add 2 mL of 1X wash buffer to each tube 10 Mix the cells 11 Centrifuge cells at lt 400 X g for 5 minutes at room temperature RT Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 19 FLICA Booklet 12 Carefully remove and discard supernatant 13 Gently vortex the cell pellet to disrupt any cell to cell clumping 14 Resuspend the cell pellet in 1 mL 1X wash buffer 15 Centrifuge cells at lt 400 X g for 5 minutes at RT 16 Carefully remove and discard supernatant 17 Gently vortex the cell pellet to disrupt any cell to cell clumping 18 Resuspend the cell pellet in 400 uL 1X wash buffer 19 At this point cells may be fixed Step 20 or analyzed directly Step 21 20 If desired fix cells by adding 40 uL fixative to the 400 uL cell suspension and mix Or if a different volume was used add the fixative at a 1 10 ratio into the volume of cell suspension to be fixed Keep fixed cells at 2 C 8 C protected from light for up to 24 hours Go on to Step 22 21 Put samples on ice 22 For single color analysis use a 15 mW argon ion laser at 488 nm Measure fluorescein on the FL1 channel Generate a log FL1 X axis versus number of cells Y axis histogram On the histogram there will appear two cell populations represe
10. s sssssensersrrnsrnrrnrnnn 20 added to a population of cells the FAM VAD FMK FLICA probe enters each 25 Flow Cytometry Sample Data cccccecceeeeeeceeeeseeneeeeeaes 22 cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer thereby inhibiting further 2D FOTIA CC Seach detecic ternbsscouemateonce deeasctesnd teats Bas teiractate Wee edema aenents wood eciadeames 23 enzymatic activity Because the FAM VAD FMK FLICA reagent becomes covalently coupled to the enzyme it is retained within the cell while any unbound FAM VAD FMK FLICA reagent will diffuse out of the cell and is Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 3 Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 4 FLICA Booklet washed away The remaining green fluorescent signal is a direct measure of the number of active caspase enzymes that were present in the cell at the time the reagent was added Cells that contain the bound FLICA can be analyzed by 96 well plate based fluorometry fluorescence microscopy or flow cytometry Because the FLICA reagent FAM VAD FMK irreversibly binds to many activated caspases caspase 1 3 4 5 6 7 8 and 9 it can be used as a generic probe for the detection of most caspases In comparison when the FLICA reagent FAM YVAD FMK enters the cell it primarily binds to caspase 1 therefore it can be used to measur
11. 5 nm for PI and if Hoechst is used a UV filter with excitation at 365 nm emission at 480 nm and slides Flow cytometer equipped with a 15 mW 488 nm argon excitation laser with appropriate filters excitation 490 nm emission gt 520 nm for FLICA excitation at 490 nm and emission at 635 nm for PI 5 Storage and Shelf Life Store the unopened kit and each unopened component at 2 C to 8 C until the expiration date Protect the FLICA reagent from light at all times Once reconstituted the 150X FLICA stock should be stored at 20 C protected from light This reagent is stable for up to 6 months and may be thawed twice during that time Once diluted store the 1X wash buffer at 2 8 C up to 14 days Replacement components can be ordered by calling ICT at 1 800 829 3194 or 952 888 8788 6 Safety Information Use gloves while handling the FLICA reagent propidium iodide Hoechst stain and fixative Dispose of all liquid components down the sink and flush with copious amounts of water Solid components may be tossed in standard trash bins MSDS sheets are available at www immunochemistry com or by calling 1 800 829 3194 or 952 888 8788 7 Overview of the FLICA Protocol Staining apoptotic cells with the FLICA kit can be completed within a few hours However the FLICA kit is used with living cells which require periodic maintenance and cultivation several days in advance In addition once the proper n
12. ce PI on the FL2 channel Generate a log FL1 X axis versus log FL2 Y axis dot plot Put in quadrant cursors The 4 quadrant areas contain the following cell populations Figure 5 i quadrant 1 PI positive fluorescein negative cells ii quadrant 2 fluorescein positive PI positive cells ili quadrant 3 fluorescein negative PI negative cells iv quadrant 4 fluorescein positive PI negative cells The cell population in quadrant 4 consists of living caspase positive cells 25 Flow Cytometry Sample Data co A B hli hit TE N 1 WwW LLI mm m Wo ole m me ene ioe ile elie eli FL1 H FL1 H Figure 5 Jurkat cells were treated with DMSO non induced cells A or camptothecin induced cells for 3 hours B Cells were labeled with FAM VAD FMK for 1 hour washed and analyzed Caspase activity was detected using a BD Facscalibor flow cytometer The frequency histogram of the number of events Y axis versus fluorescein intensity X axis shows 2 peaks caspase negative cells occur to the left of the M1 region unlabeled cells caspase positive cells lay within the M1 region cells were labeled with FLICA Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 22 FLICA Booklet 26 References 1 Slee E A C Adrain and S J Maritin 1999 Serial Killers ordering caspase activation events in apoptosis Cell Death and Differ 6 1067 1074 Walker N P R V Talanian K
13. e Microscopy Staining Protocol for Suspension Cells 1 2 10 11 12 13 Immunochemistry Technologies LLC Culture cells to a density optimal for apoptosis induction according to your specific induction protocol as discussed in Section 7 Cultivate or concentrate cells to a density of at least 5 X 10 cells mL Cell density in the cell culture flasks should not exceed 10 cells mL Cells cultivated in excess of this concentration may begin to naturally enter apoptosis Optimal cell concentration will vary depending on the cell line used Induce cells to undergo apoptosis and take samples according to your specific protocol as mentioned in Section 8 At the same time culture an equal volume of non induced cells for a negative control cell population Make sure that both the negative control and induced positive cell population tubes contain similar quantities of cells Transfer 290 300 uL of each induced and negative control cell populations into fresh tubes Or if desired larger cell volumes can be used however more of the 30X FLICA solution may be required Larger volume cell suspensions label nicely using 25 cm tissue culture flasks laid flat as incubator vessels When ready to label with the 30X FLICA solution cells should be at least 5 X 10 cells mL Density can be determined by counting cell populations on a hemocytometer Add 10 uL of the 30X working dilution FLICA solution directly to each 290
14. e the amount of active caspase 1 that was present in the cell at the time when the FAM YVAD FMK FLICA reagent was added Other green fluorescence FLICA Apoptosis Detection Kits are available to analyze specific caspases FAM VDVAD FMK FLICA can be used to detect caspase 2 FAM DEVD FMK to detect caspase 3 and caspase 7 FAM VEID FMK to detect caspase 6 FAM LETD FMkK to primarily detect caspase 8 FAM LEHD FMkK to primarily detect caspase 9 FAIM AEVD FMK to detect caspase 10 and FAM LEED FMK to detect primarily caspase 13 Following the suggested protocols listed here each sample requires 10 uL of 30X FLICA solution equal to 2 uL of 150X FLICA stock The FLICA 25 Kit will test 25 samples the FLICA 100 Kit will test 100 samples The FLICA kit was designed to evaluate apoptotic events using 3 different fluorescence detection methods 96 well microtiter plate fluorometry for quantitation fluorescence microscopy for qualitative analysis and flow cytometry for quantitation The FLICA reagent has an optimal excitation range from 488 492 nm and emission range from 515 535 nm the excitation emission pairs which best approximate this optimal range should be used Cells labeled with the FLICA reagent may be read immediately or preserved for 24 hours using the fixative Unfixed samples may be analyzed with propidium iodide or Hoechst stain Using a fluorescence plate reader with black microtiter plates apoptosis can be quantitated as t
15. ection 11 10 If desired fix cells Section 12 11 Analyze data via microtiter plate fluorometry fluorescence microscopy or flow cytometry oad RUB 8 Induction of Apoptosis The FLICA kit works with your current apoptosis protocols induce apoptosis as you normally would then label the cells with FLICA Four quick examples of protocols to induce apoptosis in suspension culture are 1 treating Jurkat cells with 2 ug ml camptothecin for 3 hours 2 treating Jurkat cells with 1 uM staurosporine for 3 hours 3 treating HL 60 cells with 4 ug ml camptothecin for 4 hours 4 treating HL 60 cells with 1 uM staurosporine for 4 hours 9 Preparation of 1X Wash Buffer The wash buffer is supplied as a 10X concentrate which must be diluted to 1X with DI H20 prior to use 1 If necessary gently warm the 10X concentrate to completely dissolve any salt crystals that may have come out of solution 2 For the FLICA 25 Kit add the entire bottle 15 mL Part 600035 of 10X wash buffer to 135 mL of DI H20 to make 150 mL 3 Or for the FLICA 100 Kit add the entire bottle 60 mL Part 600034 of 10X wash buffer to 540 mL of DI H2O to make 600 mL 4 Or if not using the entire bottle dilute the 10X wash buffer 1 10 in DI H20 For example add 10 mL 10X wash buffer to 90 mL DI H20 to make 100 mL 5 Let the solution stir for 5 minutes or until all crystals have dissolved 6 If not using the 1X wash buffer the same day it was prepared
16. g in the cell a sign it is dying The cell in the bottom left of photo B which is not visible in photo A does not have a brightly stained nucleus therefore it is not apoptotic nor necrotic Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 17 FLICA Booklet Figure 3 Suspension cells were incubated with 1 uM staurosporine for 3 hours at 37 C to induce apoptosis Cells were then labeled with FAM VAD FMK for 60 minutes at 37 C Cells were washed then Hoechst stain was added and incubated for 5 minutes Wet mount slides were prepared and 2 photos were taken of the same cells and superimposed Caspase activity green was detected using a band pass filter excitation at 488 nm emission at 520 nm Nuclear staining by Hoechst stain blue was revealed using a UV filter excitation at 365 nm emission at 480 nm In Figure 3 only one cell of the three cells appears green middle of the picture it is apoptotic and stained positive for poly caspase activity with the FAM VAD FMK reagent It also has many bright blue spots from the Hoechst stain indicating that the cell is beginning to die All the cells were stained with Hoechst The top left cell is somewhat blue throughout the cell it is not dying and is neither necrotic nor apoptotic The middle cell has many bright blue spots indicating that chromosome condensation has begun and the cell is beginning to die The bottom right cell has a very brightly s
17. he 30X working solution should be prepared immediately prior to use however the reconstituted 150X stock concentrate can be stored at 20 C for future use The newly reconstituted 150X FLICA stock must be used or frozen immediately after it is prepared and protected from light during handling 1 Reconstitute each vial of lyophilized FLICA with 50 L DMSO This yields a 150X concentrate The FLICA 25 kit contains 1 vial the FLICA 100 kit contains 4 vials 2 Mix by swirling or tilting the vial allowing the DMSO to travel around the base of the amber vial until completely dissolved At room temperature RT this reagent should be dissolved within a few minutes 3 If immediately using this solution dilute it to 30X Section 14 4 Or if using later aliquot and store it at 20 C Section 15 14 Preparation of 30X FLICA Solution for Immediate Use Using the freshly reconstituted 150X FLICA stock prepare the 30X working strength FLICA solution by diluting the stock 1 5 in PBS at pH 7 4 Following the suggested protocols here each sample to be tested requires only 10 uL of 30X FLICA solution or 2 uL of the 150X FLICA stock 1 If you are using the entire vial add 200 uL PBS pH 7 4 to each vial each vial contains 50 uL of the 150X stock this yields 250 uL of a 30X solution 2 If not using the entire vial dilute the 150X stock 1 5 in PBS pH 7 4 For example add 10 uL of the 150X stock to 40 uL PBS this yields 50
18. he amount of green fluorescence emitted from FLICA probes bound to caspases Cell populations in more advanced stages of apoptosis will have a higher RFU intensity than cell populations in earlier stages see Section 19 for sample data Viewing cells through a fluorescence microscope apoptotic cells will fluoresce green while non apoptotic cells will appear mostly unstained As apoptosis progresses the amount of active caspase enzymes capable of binding the FLICA increases and eventually reaches a maximum level Therefore cells in more advanced stages of apoptosis will appear brighter green than cells in earlier stages see Section 22 for sample data Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 5 FLICA Booklet Using a flow cytometer analysis is done using a 15 mW argon ion laser at 488 nm Fluorescein is measured on the FL1 channel and a log FL1 X axis versus number of cells Y axis histogram may be generated On this histogram there will appear two cell populations represented by two peaks The majority of the caspase negative cells will occur within the first log decade of the FL1 X axis first peak whereas the caspase positive cell population will appear as a separate peak or as a shoulder of the first peak showing increased fluorescence intensity Cells with the active caspase those undergoing apoptosis will fluoresce green See Sections 25 and 26 for sample data For Research Use Only Not f
19. i Immunochemistry Technologies LLC hod Carboxyfluorescein FLICA Apoptosis Detection K t Caspase Assay Poly Caspases FLICA FAM VAD FMk Caspase 1 FLICA FAM YVAD FMk Caspase 2 FLICA FAM VDVAD FMk Caspase 3 FLICA FAM DEVD FMK Caspase 6 FLICA FAM VEID FMK Caspase 8 FLICA FAM LETD FMK Caspase 9 FLICA FAM LEHD FMK Caspase 10 FLICA FAM AEVD FMK Caspase 13 FLICA FAM LEED FMK mn IN NN aa For Research Use Only Copyright 2002 Immunochemistry Technologies LLC FLICA Booklet For technical questions and orders please contact us at 1 800 829 3194 952 888 8788 952 888 8988 fax www immunochemistry com FLICA Apoptosis Detection Kit Ordering Information Caspase FLICA Peptide 25 test kit 100 test kit Poly Caspases FAM VAD FMK part 91 part 92 Caspase 1 FAM YVAD FMK part 97 part 98 Caspase 2 FAM VDVAD FMK part 918 part 919 Caspases 3 amp 7 FAM DEVD FMK part 93 part 94 Caspase 6 FAM VEID FMK part 95 part 96 Caspase 8 FAM LETD FMK _ part 99 part 910 Caspase 9 FAM LEHD FMK part 912 part 913 Caspase 10 FAM AEVD FMK part 922 part 923 Caspase 13 FAM LEED FMK _ part 929 part 930 Poly Caspases SR VAD FMK part 916 part 917 Caspase 3 amp 7 SR DEVD FMK part 931 part 932 Publication Version FAM FLICA Flat Manual 818 081502 No part of this manual may be reprinted without the express written permission of Immunochemistry Technologies LLC www immunochemistry com Immunochemistry Techn
20. ide of the graph for 2 hours at 37 C Cells were labeled with FAM VAD FMK solution for 60 minutes at 37 C Samples were read on a 96 well fluorescence plate reader Molecular Devices Gemini XS set at 490 nm excitation and 520 nm emission using a 495 nm cut off filter As the caspases became more active indicating apoptosis the amount of green fluorescence increased by over 500 in the induced Jurkat cells 1 800 829 3194 www immunochemistry com 13 FLICA Booklet 20 Fluorescence Microscopy Staining Protocol for Adherent Cells 1 2 3 4 ee a 11 12 13 14 15 Immunochemistry Technologies LLC Trypsinize cells Count cells Seed about 10 10 cells onto a sterile glass coverslip in a 35 mm petri dish or onto chamber slides Grow cells in their respective cell culture media formulation for 24 hours at 37 C as discussed in Section 7 Induce cells to undergo apoptosis and sample at time points according to your specific protocol as mentioned in Section 8 Add the 30X FLICA solution to the medium at a 1 30 ratio For example add 10 ul 30X FLICA to 290 300 ul medium Each investigator should adjust the amount of FLICA reagent used to accommodate their particular cell line and research conditions Mix well Incubate cells for 1 hour at 37 C under 5 COs Remove the medium If cells are to be monitored using Hoechst stain add 1 5 uL Hoechst stain to 300 uL media 0 5 v v Add this
21. immunochemistry com Immunochemistry Technologies LLC 9401 James Avenue South Suite 155 Bloomington MN 55431 t 800 829 3194 952 888 8788 fax 952 888 8988 Copyright 2002 Immunochemistry Technologies LLC
22. ion from a Frozen Aliquot 11 FLICA Apoptosis Detection Kits use a novel approach to detect active E caspases The methodology is based on a Fluorochrome Inhibitor of 17 96 Well Fluorescence Plate Reader Staining Protocol 11 Caspases FLICA Once inside the cell the FLICA inhibitor binds 18 96 Well Fluorescence Plate Reader Set UD ccsccssecseeseeseee 13 covalently to the active caspase These inhibitors are cell permeable and non cytotoxic For kits using green fluorescence a carboxyfluorescein 19 96 Well Fluorescence Plate Reader Sample Data Seis Seem E EEE dave eevee 13 labeled fluoromethyl ketone peptide inhibitor of caspases is used ICT also offers a line of red FLICA Apoptosis Detection Kits that use sulforhodamine 20 Fluorescence Microscopy Staining Protocol for Adherent Cells 14 ee l labeled inhibitors please contact ICT for more details 21 Fluorescence Microscopy Staining Protocol for Suspension Cells 15 For example the Poly Caspases FLICA Apoptosis Detection Kit contains 22 Fluorescence Microscopy Sample Data ccccceceeeee eens 17 ICT s green fluorescent labeled inhibitor FAIM VAD FMK which is a 23 Flow Cytometry with Single Color Staining cccceeeee eee 19 carboxyfluorescein FAM derivative of valylalanylaspartic acid VAD a fluoromethyl ketone FMK a potent inhibitor of caspase activity When 24 Flow Cytometry with Bicolor Staining
23. media to the cells a Incubate for 5 minutes at 37 C under 5 COs b Goon to Step 11 Wash cells twice with 2 ml 1X wash buffer At this point cells may be analyzed directly Step 13 or fixed and analyzed later Step 14 To analyze directly mount a coverslip with cells facing down onto a microscope slide containing a drop of 1X wash buffer Or remove the plastic frame of the chamber slide add a drop of 1X wash buffer onto the glass slide and cover with a coverslip Go on to Step 15 To fix the cells and analyze later add fixative to wash buffer at a 1 10 ratio For example add 40 uL fixative to 360 uL 1X wash buffer a Mount a coverslip with cells facing down onto a microscope slide containing a drop of fixative plus wash buffer Or remove the plastic frame of the chamber slide add a drop of fixative plus wash buffer onto the glass slide and cover with a coverslip b Keep fixed cells at 2 C 8 C protected from light for up to 24 hours Go on to Step 15 Observe cells under a fluorescence microscope using a bandpass filter excitation 490 nm emission gt 520 nm to view the green fluorescence of caspase positive cells If Hoechst stain was also used it can be seen using a UV filter with excitation at 365 nm and emission at 480 nm If these filters are not available select a filter combination that best approximates these settings 1 800 829 3194 www immunochemistry com 14 FLICA Booklet 21 Fluorescenc
24. minutes at RT 16 Carefully remove and discard supernatant 17 Gently vortex the cell pellet to disrupt any cell to cell clumping 18 Resuspend the cell pellet in 1 mL 1X wash buffer 19 Determine the concentration of both the induced and non induced cell populations This can be done while the cells are being pelleted down for the last time Step 20 To count cells a Remove 50 uL from each tube b Add to 450 uL PBS forming a 1 10 dilution of each c Count the cells using a hemocytometer d After counting compare the density of each The non induced population may have more cells than the induced population as some induced cells may be lost during the apoptotic process If there is a dramatic loss in stimulated cell population numbers adjust the volume of the induced cell suspension to match the cell density of the non induced suspension Step 24 20 Centrifuge the remaining cells at lt 400 X g for 5 minutes at RT 21 Carefully remove and discard supernatant 22 Resuspend non stimulated cells in 400 uL PBS 23 If it is not necessary to equilibrate the cell concentrations as discussed in Step 19d resuspend the stimulated cells in 400 uL PBS as well 24 If it is necessary to equilibrate the cell concentrations from Step 19d adjust the suspension volume of the PBS for the induced cells to approximate the cell density of the non induced population This Immunochemistry Technologies LLC 1 800 829 3194 www immunoche
25. mistry com 12 25 26 FLICA Booklet adjustment step is optional if your cell treatment does not result in a dramatic loss in stimulated cell population numbers Place 100 uL of the cell suspensions into each of 2 wells of a black microtiter plate Do not use clear plates Avoid bubbles Measure the fluorescence intensity of fluorescein Section 18 18 96 Well Fluorescence Plate Reader Set Up 1 2 Set the plate reader to perform an endpoint read Set the excitation wavelength at 490 nm and the emission wavelength to 520 nm Fluorescein has an optimal excitation range from 488 492 nm and emission range from 515 535 nm Select the filter pairing which most closely approximates this range the filter pairing used may differ slightly from these optimal settings Read the sample An example of differential FLICA fluorescence intensities in induced versus non induced Jurkat cells is shown in Figure 1 using a 96 well fluorescence plate reader 19 96 Well Fluorescence Plate Reader Sample Data Immunochemistry Technologies LLC Jurkat Cells FAM VAD FMk RFUs Negative Control Non induced std dev 1 58 Apoptotic Induced std dev 1 60 Figure 1 FAM VAD FMK fluorometric detection of active caspases in Jurkat cells SD of 6 wells In Figure 1 cells were either treated with DMSO negative non induced cells bar on the left side of the graph or with staurosporine apoptotic induced cells bar on the right s
26. nted by two peaks The majority of the caspase negative cells will normally occur within the first log decade of the FL1 X axis first peak whereas the caspase positive cell population will appear as a separate peak or as a shoulder of the first peak showing increased fluorescence intensity Position the vertical cursor in the gap between the two peaks Events falling to the right of the vertical cursor should be counted as caspase positive 23 See Figure 5 for an example of single color analysis using FACS 24 Flow Cytometry with Bicolor Staining If a bicolor analysis is desired cells may be stained with PI along with the FLICA reagent PI stains necrotic dead and membrane compromised cells Using PI a flow cytometer can distinguish among live cells dead cells caspase negative and caspase positive cells Positive control samples can be prepared by inducing cells in Suspension culture to undergo apoptosis see Section 8 for examples of apoptosis induction protocols After labeling with FLICA and PI cells can be analyzed directly by flow cytometry For a thorough bicolor analysis 4 types of samples are recommended to set up electronic compensation and quadrant statistics both types should have induced and non induced populations a unstained cells induced and non induced b cells stained with FLICA only induced and non induced c cells stained with PI only induced and non induced d cells stained with FLICA and
27. ologies LLC 9401 James Avenue South Suite 155 Bloomington MN 55431 rt 800 829 3194 952 888 8788 fax 952 888 8988 Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 2 FLICA Booklet FLICA Booklet Table of Contents 1 Introduction Apoptosis is an evolutionarily conserved form of cell suicide which follows a specialized cellular process The central component of this process is a cascade of proteolytic enzymes called caspases These enzymes participate in a series of reactions that are triggered in response to pro 2 Contents of the FLICA Apoptosis Detection Kit 0 0 ccc eee 6 apoptotic signals and result in the cleavage of protein substrates causing the disassembly of the cell 1 Introduction 2 0 0 cece cece cca eccecceucceuceucenueeuceeucenutenuenucenunnannnees 4 3 Recommended Materials and Equipment cccce cece eee 6 AASE TAU ON raa EA ES 7 Caspases have been identified in organisms ranging from C elegans to humans The mammalian caspases play distinct roles in apoptosis and 5 Storage and Shelf Life 0 0 cece ccc cecccceccceucceuuccuuceenaceeanes T inflammation In apoptosis caspases are responsible for proteolytic 6 Safety Information 0 c ccc cece cece cece eee e eee e ee eeaeeeeeeeeeenaeeees 7 cleavages that lead to cell disassembly effector caspases and are involved in upstream regulatory events initiator caspases An active 7 Ove
28. or use in diagnostic procedures 2 Contents of the FLICA Apoptosis Detection Kit FAM XXX FMK FLICA Reagent lyophilized 25 test kits contain 1 vial of the FLICA 100 test kits contain 4 vials 10X Wash Buffer 60 mL part 634 or 15 mL part 635 Fixative 6 mL part 636 Propidium lodide 1 mL part 638 Hoechst Stain 1 mL part 639 Assay Manual part 817 MSDS sheets 3 Recommended Materials and Equipment not all are required Cultured cells with media Reagents to induce apoptosis 15 mL polystyrene centrifuge tube 1 per sample Amber vials or polypropylene tubes for storage of 150X concentrate at 20 C if aliquoted 150 mL or 600 mL graduated cylinder Slides Hemocytometer Clinical centrifuge at lt 400 X g 37 C CO incubator Vortexer Pipette s capable of dispensing at 10uL 50uL 200uL 300uL 1mL dl H2O 135 mL or 540 mL needed Phosphate Buffered Saline PBS pH 7 4 up to 100 mL needed Dimethyl Sulfoxide DMSO 50uL or 200uL needed Ice or 4 C refrigerator to store cells 4 Instrumentation not all are required 96 well fluorescence plate reader with excitation at 488 nm emission 520 nm filter pairings and black round or flat bottom 96 well microtiter plates Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 6 FLICA Booklet Fluorescence microscope with appropriate filters excitation 490 nm emission gt 520 nm for FLICA excitation at 490 nm and emission at 63
29. protocol Section 17 20 21 23 24 or 25 17 96 Well Fluorescence Plate Reader Staining Protocol Following this fluorescence plate reader protocol each sample requires 10 uL of 30X FLICA solution equal to 2 uL of 150X FLICA stock 1 As discussed in Section 7 culture cells to a density optimal for apoptosis induction according to your specific induction protocol Cell density in the cell culture flasks should not exceed 10 cells mL Cells cultivated in excess of this concentration may begin to naturally enter apoptosis Optimal cell concentration will vary depending on the cell line used 2 Induce apoptosis following your protocol as mentioned in Section 8 3 Atthe same time culture an equal volume of non induced cells for a negative control cell population Make sure that both tubes of cells contain similar quantities of cells Cells can be concentrated just prior to induction to 2 6 X 10 cells mL Cells may be induced at even lower concentrations but must be concentrated to 1 X 10 cells mL for FLICA labeling If necessary cells can be concentrated by centrifugation for 5 minutes at lt 400 X g at RT 4 Once induction is completed transfer 290 300 uL of each cell suspension to sterile tubes Larger cell volumes can also be used as determined by each investigator however more of the FLICA reagent may be needed per sample Larger volume cell Suspensions label nicely using 25 cm tissue culture flasks laid
30. reen If Hoechst stain was also used it can be seen using a UV filter with excitation at 365 nm and emission at 480 nm If these filters are not available select a filter combination that best approximates these settings Examples of FLICA and Hoechst staining of Jurkat cells are shown in Figures 2 3 and 4 1 800 829 3194 www immunochemistry com 16 FLICA Booklet 22 Fluorescence Microscopy Sample Data E Figure 2 Suspension cells were incubated with 1 uM staurosporine for 3 hours at 37 C to induce apoptosis Cells were then labeled with FAM VAD FMK for 60 minutes at 37 C Cells were washed then Hoechst stain was added and incubated for 5 minutes Wet mount slides were prepared and 2 photos were taken of the same cells Caspase activity on left photo A was detected using a band pass filter excitation at 488 nm emission at 520 nm Nuclear staining by Hoechst stain on right photo B was revealed using a UV filter excitation at 365 nm emission at 480 nm In Figure 2 photo A only one cell appears green it is apoptotic and stained positive for poly caspase activity with the FAM VAD FMK reagent The other cell which is not visible did not bind to the reagent and therefore is not apoptotic The same cells photographed at right under a different wavelength for Hoechst stain appear blue The cell in the top right of photo B which appears green in photo A has a very brightly stained nucleus its DNA is condensin
31. rview of the FLICA Protocol cccceeeeeeeeeeeeeeeeeeneeees 7 caspase consists of two large 20 kD and two small 10 kD subunits that form two heterodimers which associate in a tetramer In common with S INGUGHON Of ADODIOSIS cies acccdeecectence senapenassbussantacsiedeuceeswaseuais 8 other proteases caspases are synthesized as precursors that undergo 9 Preparation of 1X Wash Buffer 0 0 00c c cece ec ecececee ee ee eens eee 8 proteolytic maturation either autocatalytically or in a cascade by enzymes ith similar specificity 10 Propidium lodide cccccccccececessececeeseseeseseeseseeesesevsesevsteeeee 9 E ie ee Hoe che a oaeee E E T 9 Caspase enzymes specifically recognize a 4 amino acid sequence on the aa target substrate which necessarily includes an aspartic acid residue This 12 Fixative E EEE A EE EEEE E E E E E eee eee eke ET 9 residue is the target for the cleavage reaction which occurs at the carbonyl 13 Reconstitution of the 150X FLICA Stock ccc cece cece e eee ee 10 end of the aspartic acid residue Caspases can be detected via immuno precipitation immunoblotting techniques using caspase specific antibodies 14 Preparation of 30X FLICA Solution for Immediate Use 10 or by employing fluorochrome substrates which become fluorescent upon 15 Storage of 150X FLICA Stock for Future USe eccccccccccecceceseseeeees 10 Epavagen yg CA RASE 16 Preparation of 30X FLICA Solut
32. specific induction protocol 2 Induce apoptosis following your protocol as mentioned in Section 8 3 Culture an equal volume of non induced cells for a negative control cell population Make sure that both tubes of cells contain similar quantities of cells Ideally cells should be at 1 X 10 cells mL for FLICA labeling If necessary cells may be induced to undergo apoptosis at lower concentrations but then concentrated just prior to labeling to 0 5 2 X 10 cells mL by centrifugation for 5 minutes at lt 400 X g at RT 4 Once induction is completed transfer 300 uL of each cell suspension to Sterile tubes Larger cell volumes can also be used as determined by each investigator however more of the FLICA reagent may be needed per sample Larger volume cell suspensions label nicely using 25 cm tissue culture flasks laid flat as the incubation vessel 5 Add 10 uL 30X FLICA solution directly to the 300 uL cell suspension 6 Or if a different cell volume was used add the 30X FLICA solution at a 1 30 ratio For example if 2 9 mL of cell suspension was used add 100 uL of the 30X FLICA solution forming a final volume of 3 mL Each investigator should adjust the amount of FLICA reagent used to accommodate their particular cell line and research conditions 7 Mix the cells by slightly flicking the tubes 8 Incubate cells for 1 hour at 37 C under 5 COs protecting the tubes from light As cells may settle on the bottom
33. tained blue nucleus indicating that it is dying This cell did not stain green so it is either necrotic caspases were not activated and therefore could not bind to FAM VAD FMk or apoptotic but far past the active caspase stage and therefore did not stain green with FAM VAD FMK because the caspases were no longer active Figure 4 Cells were prepared as in Figure 3 and photos superimposed Both cells are apoptotic green and dying blue nuclei As the cell at left is much brighter green than the right cell the left cell had more active caspases Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 18 FLICA Booklet 23 Flow Cytometry with Single Color Staining Positive control samples can be prepared by inducing cells in suspension culture to undergo apoptosis see Section 8 for examples of apoptosis induction protocols Following this flow cytometer protocol each sample requires 10 uL of 30X FLICA solution equal to 2 uL of 150X FLICA stock After labeling with FLICA cells can be analyzed directly by flow cytometry or the cells may be fixed first and then analyzed by flow cytometry Fora thorough analysis 2 types of samples are recommended both should have induced and non induced populations 1 unstained cells induced and non induced 2 cells stained with FLICA induced and non induced 1 As discussed in Section 7 culture cells to a density optimal for apoptosis induction according to your
34. uL of a 30X solution Store the unused 150X stock at 20 C Section 15 3 Mix by inverting or vortexing the vial at RT The 30X working strength FLICA solution must be used the same day that it is prepared 15 Storage of 150X FLICA Stock for Future Use If not all of the 150X FLICA stock will be used the same time it is reconstituted the unused portion may be stored at 20 C for 6 months During that time the 150X FLICA stock may be thawed and used twice After the second thaw discard any remaining 150X FLICA stock If you anticipate using it more than twice make small aliquots in amber vials or polypropylene tubes and store at 20 C protected from light When ready to use follow Section 16 below Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 10 FLICA Booklet 16 Preparation of 30X FLICA Solution from a Frozen Aliquot If some of the 150X FLICA reagent was previously reconstituted and then stored at 20 C it may be used 2 more times within 6 months 1 Thaw the 150X FLICA stock and protect from light Once the aliquot has become liquid dilute the 150X stock solution 1 5 in PBS pH 7 4 For example mix 10 uL of 150X FLICA reagent with 40 uL of PBS 3 Mix by inverting or vortexing the vial at RT 4 Ifthe 150X FLICA stock was frozen immediately after reconstitution and was never thawed return it to the freezer If the stock was thawed once before discard it 5 Proceed to the labeling
35. umber of cells has been cultivated time must be allotted for the induction process which typically requires a 2 4 hour incubation at 37 C Therefore as the 30X FLICA solution must be used immediately the FLICA reagents should be prepared at the end of the apoptosis induction process The following is a quick overview of the FLICA protocol 1 Culture cells to a density optimal for apoptosis induction according to your specific induction protocol but not to exceed 10 cells mL 2 Atthe same time culture a non induced negative control cell population at the same density as the induced population for every labeling condition For example if labeling with FLICA and Hoechst stain make 8 populations a Unlabeled induced and non induced populations b FLICA labeled induced and non induced populations Immunochemistry Technologies LLC 1 800 829 3194 www immunochemistry com 7 FLICA Booklet c FLICA and Hoechst labeled induced and non induced populations d Hoechst labeled induced and non induced populations Induce apoptosis following your protocol 4 sample protocols are mentioned in Section 8 Prepare 1X wash buffer Section 9 Prepare 150X FLICA stock Section 13 Prepare 30X FLICA solution Section 14 or 16 Stain cells with 30X FLICA solution incubate for 1 hour and wash cells Section 17 20 21 23 or 24 8 If desired label cells with propidium iodide Section 10 If desired label cells with Hoechst stain S
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