The FLICA (Fluorescently Labeled Caspase Inhibitor) Caspase 3 and 7 Assay is a simple but accurate assay to measure apoptosis through caspase 3 activity in whole living cells. Four sample protocols are described below.
Grow your cells up to 1 x 10 ^ 6 cells / mL. Follow the experimental protocol where the caspase activity will be investigated; create positive and negative controls for caspase activity. Reconstitute the reagent with 50 ul DMSO to form the stock concentrate (can be frozen for future use). Dilute the stock concentrate with 200 ul of 1X PBS to form the working solution.
Add ~ 10 ul of the working solution directly to a 300-500 ul aliquot of your cell culture for labeling. Incubate 30 minutes -1 hour. Wash and centrifuge cells two to three times, or incubate for 1 hour with fresh medium or 1x apoptosis wash buffer. If desired, label cells with Hoechst stain. If desired, label cells with propidium iodide or 7-AAD. If you want, arrange the cells. Analyze the data with a fluorescence microscope, plate reader, or flow cytometer.
Prepare frozen tissues according to the experiment. Allow the slides to air dry. Fix the slides in acetone for 1 minute. Rehydrate the slides by washing (twice for 5 min) in TBS-tween (TBSt) or PBS-tween (PBS). Block slides for 20 minutes (such as 20% Aquablock on 0.2% interpolated media). Dilute 150X FLICA stock 1:50 in PBS to form a 3X working solution.
For example, add 50 uL of 150X broth to 2450 uL of PBS (2.5 mL total). Add 50 uL of 3X FLICA and incubate for> 1 h protected from light. Wash with TBSt or PBS (twice for 5 min) by placing the slides in a slide incubation dish containing 1X wash buffer. Develop with DAPI and coverslips. Store samples at 2-8 degrees C for short-term storage, staining will last at -20 degrees C for extended periods.
Adherent cells should be washed carefully to avoid loss of cells that round off the plate surface. Loose cells can be collected from the plate or the surface of the slide and treated as cells in suspension, while those that remain adhering to the surface should be washed as adherent cells. If the adherent cells are trypsinized, the loose cells can recombine with the trypsinized pool, or the washed loose cells can then recombine with the adherent part when testing is performed.
If adherent cells are grown on a tissue culture plate, the entire plate can be gently rotated as part of the washing process to pellet loose floating cells. Avoid any attempt to trypsinize cells before labeling them with a vital stain such as PI. Cell membranes exposed to trypsin could become transiently permeable to vital dyes over a variable period of time, depending on the cell line. Cells can be labeled with FLICA before or after trypsinization.
Adherent cells: trypsinization before FLICA labeling and FACS analysis
Grow cells in T25 flasks and expose them to experimental conditions. Apoptotic cells can break off and begin to float in the middle. Save and spin to pellet and include these cells in your analysis. Trypsinize adherent cells; neutralize with trypsin inhibitor present in 20% FBS cell culture media; group the cells with the granules created in n. 2; add a few ml of medium. Spin ~ 5 min at 220 x g and remove all supernatant minus ~ 100 uL. Count the cells and adjust the volume of the cell suspension to suit the experiment (typically 300-500 uL). Transfer cells to a 15 ml tube.
Add 10 – 17 uL of 30X FLICA. Incubate at 37 degrees C, 30-60 minutes, mixing gently every 10 minutes. Wash by adding ~ 10 ml of medium and incubate at 37 degrees C for 60 minutes to allow any unbound FLICA to diffuse out of the cells. Spin at 220 x g for 5 minutes; aspirate the supernatant. Add ~ 300 ul 1X Apoptosis Wash Buffer. Put cells on ice and protect them from light. If desired, add 30 uL of fixative. Analyze the cells with a flow cytometer.
Adherent cells: FLICA tag before trypsinization and FACS analysis
Seed 5-8 x 10 4 cells in a 24-well plate in a final volume of 600 uL and allow to adhere for 24 hours. Expose cells to experimental conditions. Add 1-4 uL of FLICA 150X Stock Concentrate and incubate 1-3 hours at 37 degrees C. Remove the supernatant containing the rounded cells and reserve in a labeled tube. Wash the adherent cell monolayer by gently adding PBS to cover the adherent cell monolayer.
Remove the PBS and combine it with the cells previously reserved in step 4. Add trypsin-versene to just barely cover the attached cell monolayer. Allow cells to detach and remove detached cells by adding 1 ml of cell culture medium + 20% FBS to the trypsinized cells in the wells. Add detached cells from the trypsinization step to the supernatant from step 4. Add 2 ml of cell culture medium + 20% FBS to each tube containing trypsinized cells. Spin the cells at 220 x g for 5 min.
Remove the supernatant and discard it. Add 1 ml of 1x Apoptosis Wash Buffer. Spin the cells at 220 x g for 5 min. Remove the supernatant. Add 1 ml of 1x Apoptosis Wash Buffer. Spin the cells at 220 x g for 5 min. Remove the supernatant and resuspend in 300 uL of 1X apoptosis wash buffer. If desired, add 30 ul of fixative. Analyze in FACS immediately.