CASPASE APOPTOSIS PROTOCOLS FOR FLICA DETECTION KITS FOR IN VITRO USE

Protocols for FLICA™ Apoptosis Detection Kits
Detect caspase activity in whole living cells 

See our main FLICA™ page for product details

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Read the green FAM manual
Read the red SR manual
Caspases discovered in yeast - read the FACS protocol (SR-FLICA™)

Suspension Cells with FAM-FLICA™ (green fluorescence)

1. Culture your cells up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol, and create positive and negative controls. 
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which 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. You may need to use more or less reagent than we suggest.
6. Incubate 1-4 hours. The reagent should start to react with the caspases within 15 minutes.
7. Spin cells ~5 minutes at 220g and carefully remove the FLICA™-labeled supernatant.
8. Add at least 400uL of the FLICA™ 1x apoptosis wash buffer (or use cell culture media).
9. Let the buffer incubate with the cells for 5-15 minutes protected from light (cells may be placed in the incubator).  
10. Carefully aspirate the buffer and discard.
11. Wash the cells up to 3 times by repeating Steps 7-10.  After the final wash, resuspend the cells in 1x apoptosis wash buffer for analysis. Some cells may need to be washed more than others depending on the type of instrument used.  Cells evaluated by flow cytometry may not need to be washed as much as cells evaluated in a plate reader or microscope, as the sheathing fluid acts as a wash buffer.  Cells analyzed in a plate reader often have to be washed the most, as the fluorometer measures total fluorescence within the well and any excess reagent will lead to higher RFUs. 
12. If desired, label cells with Hoechst stain.
13. If desired, label cells with Propidium Iodide or 7AAD.
14. If desired, fix cells. 
15. Analyze data using a fluorescence microscope, plate reader, or flow cytometer.

Require wash step.

Good for end-point experiments.

Cells fluoresce until cell membrane breaks down.

Read with fluorescence microscope, plate reader, or flow cytometer.  

FAM-FLICA™ (green) excites at 490 and emits at 520 nm.

FAM-FLICA™ is compatible with DAPI, SR-FLICA, Hoechst, PI, 7-AAD, and other fluorescent reagents for dual-staining studies.

Adherent Cells with FAM-FLICA™ (green fluorescence)

Read the FAM manual

Adherent cells need to be carefully washed to avoid the loss of any cells which round up and come off the plate surface. Loose cells may be harvested from the plate or slide surface and treated as suspension cells, while those remaining adherent to the surface should be washed as adherent cells. If the adherent cells are trypsinized, the loose cells can be recombined with the trypsinzed pool, or the washed loose cells can then be recombined with the adherent portion when the analysis is performed. If growing adherent cells in a plate, the entire plate may be gently spun as part of the wash process to sink any loose floating cells (however this may be harmful to some cell lines so you may want to try several wash techniques). Cells may be labeled with FLICA™ before or after trypsinization. Here are 2 protocols for adherent cells.

Sample protocol for adherent cells, trypsinize first, then label with FLICA™, FACS analysis:
1. Culture your cells in T25 flasks. 
2. Induce apoptosis following your protocol, and create positive and negative controls. Apoptotic cells may detach begin to float into the media. Save and spin to pellet and include these cells in your analysis #7. 
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
4. Dilute the stock concentrate with 200uL 1X PBS to form the working solution. 
5. Trypsinize adherent cells, neutralize with trypsin inhibitor, and pool cells with any pellets created in #2, add a few mL media.
6. Spin cells ~5 minutes at 220g and remove all but ~100mL supernatant.
7. Count cells and adjust volume of cell suspension to fit your experiment (typically 300-500ul), transfer cells into a 15mL tube.
8. Add 2-10uL of the FLICA working solution to the 300-500uL aliquot of cells. 
9. Incubate at 37C, 30-60 minutes, mixing gently every 10 minutes.
10. Wash by adding ~10mL media to each tube and incubate at 37C for 60 minutes to allow any unbound FLICA™ to diffuse out of the cells.
11. Spin cells at 220g for 5 minutes, aspirate supernatant.
12. Add ~300uL 1X Apoptosis Wash Buffer. Keep cells on ice, protected from light until analysis. 
13. If desired, also add 30uL fixative.
14. Analyze samples on a flow cytometer.

Sample protocol for adherent cells, label first with FLICA™, then trypsinize, FACS analysis:
1. Seed 50-80000 cells in a 24-well plate in a final volume of 600 mL and let them attach for 24 hours.
2. Induce apoptosis.
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which can be frozen for future use). 
3. Added 1 to 4 uL of FLICA™ 150X stock concentrate to each well and incubate 1 to 3 hours,  at 37C. 
4. Remove supernatant
5. Wash cells with PBS and add to supernatant
6. Trypsinize the attached cells.
7. Add detached cells to the pool of supernatant +PBS wash
8. Add 2 ml of 1x apoptosis wash buffer
9. Spin cells 220g for 5 min and remove supernatant (discard)
10. Add 1mL 1x apoptosis wash buffer and spin again.
11. Remove supernatant and resuspend in 300  mL of wash buffer. 
12. Analyze on FACS immediately.

Suspension Cells with SR-FLICA™ (red fluorescence)

Read the SR manual

Caspases discovered in yeast - read the FACS protocol

1. Culture your cells up to 1 x 10⁶ cells/mL. 
2. Induce apoptosis following your protocol, and create positive and negative controls. 
3. Reconstitute the reagent with 50uL DMSO to form the stock concentrate (which 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 1-4 hours. 
7. Wash and spin cells twice, 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, plate reader, or flow cytometer.

SR-FLICA™ (red) excites at 560 and emits at 600 nm.

SR FLICA™ is compatible with DAPI, FAM-FLICA, Hoechst, 7-AAD, GFP, and other fluorescent reagents for dual-staining studies.

This page was updated on 02/15/2007 . Please contact Sally Hed with questions 952-888-8788 x10 sally@immunochemistry.com .