Detect active caspase enzymes with the FAM-FLICA Poly Caspase Assay Kit. This in vitro assay employs the fluorescent inhibitor probe FAM-VAD-FMK to label active caspase enzymes in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

Caspases play important roles in apoptosis and inflammation. ICT’s FLICA assay kits are used by researchers seeking to quantitate apoptosis via caspase activity in cultured cells and tissues. The FAM FLICA Poly Caspase probe allows researchers to assess caspase activation.

The FLICA reagent FAM-VAD-FMK enters each cell and irreversibly binds to activated caspases. Because the FAM-VAD-FMK FLICA reagent becomes covalently coupled to the active enzymes, it is retained within the cell, while any unbound FAM-VAD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active caspase enzyme activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by a fluorescence plate reader, fluorescence microscopy or flow cytometry. Cells labeled with the FLICA reagent may be read immediately or preserved for 16 hours using the fixative included in the kit. Unfixed samples may also be analyzed with Propidium Iodide or Hoechst 33342 to detect changes in necrosis or nuclear morphology, respectively. Prepare samples and controls 2.

Dilute 10X Apoptosis Wash Buffer 1:10 with diH 20. Reconstitute FLICA with 50 µL DMSO. Dilute FLICA 1:5 by adding 200 µL PBS.

Add diluted FLICA to each sample at 1:30 (e.g., add 10 µL to 290 µL of cultured cells). Incubate approximately 1 hour. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells. If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.

If desired, fix cells. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLICA excites at 492 nm and emits at 520 nm. If working with adherent cells, please see the manual for additional protocols. Jurkat cells dually stained with Hoechst and poly-caspases FLICA (cat.

Caspase activity is revealed by green fluorescence in the middle cell, indicating that only this cell is apoptotic. Cell at left is also dying (scattered blue), but is not apoptotic because it is not green. Cell in upper right is healthy (concentrated blue nucleus, no green FLICA). Jurkat cells were labeled with ICT’s Poly-Caspases FLICA kit (cat. 4 cells fluoresce green (left), but the grey image (right) reveals 5 cells in the field.

The 4 green cells are apoptotic = 80% of cells in this experiment had active caspases. Data courtesy of Dr. Chen YL, Xu G, Liang X, Wei J, Luo J, Chen GN, Yan XD, Wen XP, Zhong M, Lv X. Inhibition of hepatic cells pyroptosis attenuates CLP-induced acute liver injury. Am J Transl Res. “The cells were stained with FLICA (FAM-VAD-FMK655, ImmunoChemistry Technologies) for 40 min at 37°C, followed by the addition of 7AAD (BD Pharmingen™) before loading.

The stained cells were analyzed with a FACS Calibur flow cytometer and the data with FlowJo software.” Lin JX, Du N, Li P, Kazemian M, Gebregiorgis T, Spolski R, Leonard WJ. Critical functions for STAT5 tetramers in the maturation and survival of natural killer cells.

Nov 6;8(1):1320. Doi: 10.1038/s41467-017-01477-5. “To measure caspase activity in the cells, total splenocytes were incubated at 37 °C for 1 h with fluorescent-labelled caspase inhibitor FAM-VAD-FMK probes (FLICA reagent) for Poly caspases (91), caspase-1 (97), caspases-3, 7 (93), and caspase-9 (912) according to the manufacturer’s instructions (Immunochemistry Technologies, Bloomington, MN, USA). Data were acquired using a FACSCanto II flow cytometer (BD Immunocytometry Systems) and analysed using FlowJo (v9.7.5, Tree Star, Inc., Ashland, OR).”.