1. Culture your cells up to 1 x 10^6 cells/mL.
2. Follow experimental protocol where caspase activity will be investigated; create positive and negative controls for caspase activity.
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (can be frozen for future use).
4. Dilute the stock concentrate with 200uL 1X PBS to form the working solution.
5. Add ~10uL of the working solution directly to a 300-500uL aliquot of your cell culture for labeling.
6. Incubate 30 minutes -1 hour.
7. Wash and spin cells two or three times, or let incubate for 1 hour with fresh media or 1x apoptosis wash buffer.
8. If desired, label cells with Hoechst stain.
9. If desired, fix cells.
10. Analyze data using a fluorescence microscope or fluorescence plate reader.
Tissue Sections (frozen or fresh)
1. Prepare tissue sections according to the experiment. Do not paraffin-embed or fix. Allow frozen sections to thaw before FLICA labeling.
2. Reconstitute lyophilized FLICA reagent with 50 uL DMSO to form a stock concentrate.
3. Dilute 10X Apoptosis Wash Buffer (in kit) to 1X.
4. Just prior to staining the samples, dilute the stock concentrate 1:50 in PBS to form the FLICA tissue staining working solution.
5. Add enough FLICA working solution to cover the sample.
6. Incubate at least 1 hour, protected from light.
7. Wash with TBSt, PBSt, or 1X Apoptosis Wash Buffer twice for 5-10 minutes.
8. Place slides in incubation dish containing 1X Apoptosis Wash Buffer
9. Stain nuclei with DAPI and apply coverslip.
10. Store samples at 2-8 degrees C for short term storage or at -20C for long-term storage
Adherent Cells: Trypsinization prior to FLICA labeling
1. Culture cells in T25 flasks and expose to the experimental conditions.
2. Apoptotic cells may detach and begin to float into the media. Save and spin to pellet and include these cells in your analysis.
3. Trypsinize adherent cells; neutralize with trypsin inhibitor present in 20% FBS-cell culture media; pool cells with any pellets created in #2; add a few mL media.
4. Spin ~5 minutes at 220 x g and remove all but ~100 uL supernatant.
5. Count cells and adjust volume of cell suspension to fit the experiment (typically 300-500 uL). Transfer cells into a 15 mL tube.
6. Add 10 - 17 uL of 30X FLICA.
7. Incubate at 37C, 30-60 minutes, mixing gently every 10 minutes.
8. Wash by adding ~10mL media and incubate at 37C for 60 minutes to allow any unbound FLICA to diffuse out of the cells.
9. Spin at 220 x g for 5 minutes; aspirate supernatant.
10. Add ~300uL 1X apoptosis wash buffer. Put cells on ice, and protect from light.
11. If desired, add 30 uL fixative.
12. Analyze cells via fluorescence microscopy, fluorescence plate reader, or flow cytometry (yellow or green excitation laser required for appropriate excitation of the sulforhodamine B dye label).
Adherent Cells: FLICA label prior to trypsinizing, and FACS analysis
1. Seed 5-8 x 10^4 cells in a 24-well plate in a final volume of 600 uL and let attach for 24 hours.
2. Expose cells to the experimental conditions.
3. Add 1-4 uL of FLICA 150X stock concentrate and incubate 1-3 hours at 37C.
4. Remove supernatant containing any rounded up cells and set aside in labeled tube.
5. Wash adherent cell monolayer by gently adding PBS to cover the adherent cell monolayer.
6. Remove PBS and combine with cells previously set aside in step 4.
7. Add trypsin - versene to barely cover the attached cell monolayer.
8. Allow cells to detach and remove detached cells by adding 1 mL of cell culture media + 20% FBS to the trypsinized cells in the wells.
9. Add detached cells from the trypsinization step to supernatant from step 4.
10. Add 2 mL of cell culture media + 20% FBS to each tube containing trypsinized cells.
11. Spin cells at 220 x g for 5 min. Remove supernatant and discard. Add 1mL 1x apoptosis wash buffer.
12. Spin cells at 220 x g for 5 min. Remove supernatant. Add 1mL 1x apoptosis wash buffer.
13. Spin cells at 220 x g for 5 min. Remove supernatant and resuspend in 300 uL 1X apoptosis wash buffer.
14. If desired, add 30uL fixative.
15. Analyze cells via fluorescence microscopy, fluorescence plate reader, or flow cytometry (yellow or green excitation laser required for appropriate excitation of the sulforhodamine B dye label).