Necrosis vs Apoptosis Assay Kit

1. Prepare samples and controls, 2. Dilute Apoptosis Wash Buffer 1:10 with diH2O., 3. Reconstitute FAM-VAD-FMK with 50 µL DMSO., 4. Dilute FLICA 1:5 by adding 200 µL PBS., 5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 µL of diluted FLICA to 290 µL of sample)., 6. Incubate approximately 1 hour., 7. Remove media, then wash cells: add 1X Apoptosis Wash Buffer and spin cells (twice)., 8. Resuspend samples in 400 µL 1X Apoptosis Wash Buffer, 9. Reconstitute 7-AAD with 260 µL DMSO, 10. Add 7-AAD to each sample at 1:200 (e.g., add 2 µL to 400 µL of sample), 11. Analyze with a fluorescence microscope or flow cytometer. FAM-VAD-FMK excites at 492 nm and emits at 529 nm. 7-AAD excites at 546 nm and emits at 647 nm.

Kit 9147: 50-100 tests, FAM-FLICA Poly Caspase Reagent (FAM-VAD-FMK), 2 vials, #637, 7-Aminoactinomycin D (7-AAD) vital dye, 1 0.26 mg vial, #6163, 10X Apoptosis Wash Buffer, 1 60 mL bottle, #634, Fixative, 6 mL, #636, Kit Manual

Assessment of cellular cytotoxicity levels associated with cytolytic activity of T lymphocytes or natural killer cells is an important aspect of immunobiology related research. Early assays developed to assess cytolytic activity utilized the release of 51Cr from membrane compromised target cells, which were passively loaded with this radioactive indicator prior to exposure to the cytotoxic agent or cells. A major draw-back to these 51Cr release assays is their reliance on the use of a radioactive isotope with its associated disposal and safe handling issues. Additionally, chromium uptake methods suffer from variabilities in target cell preloading inconsistencies as Well the tendency to spontaneously release the chromium isotope into the media in the absence of any cytotoxicity stimulus. Assessing potential cytotoxicity properties of chemical and biological agents is a mandatory requirement for the safe distribution of pharmaceuticals, vaccines, or additives associated with food product formulations. Early identification of unintended drug, vaccine, or chemical associated cytotoxicity properties is always an early priority of initial FDA approval testing protocols. With cellular cytotoxicity assessment playing a central role in countless research and environmental safety studies, there is an ever present need for simple, straightforward analysis methods like the Necrosis vs Apoptosis Assay developed by Immunochemistry Technologies, LLC (ICT). ICT’s Necrosis vs Apoptosis Assay simultaneously detects both apoptosis associated cytotoxicity events as Well as cell death due to necrosis. Apoptotic cells are identified using ICT’s Fluorescent Labeled Inhibitor of CAspases (FLICA®) reagent probe. The FAM-FLICA® probe covalently binds to active caspase enzymes, which are up-regulated during apoptosis, thus clearly labeling apoptotic cells for subsequent analysis. Non-apoptotic cells will not contain the active caspase enzymes required for FAM-FLICA® to remain covalently bound within the cell structure. Loss of the integrity of the cell membrane, indicative of necrosis or late stage apoptosis, is detected using the vital staining dye, 7-aminoactinomycin D (7-AAD), a red fluorescing live/dead stain. This dye easily penetrates cell membrane-compromised cells, binding tightly to GC rich regions of the DNA. 7-AAD will not label cells in early stages of apoptosis, as their cell membranes are still intact and are capable of excluding 7-AAD. As caspases are active during early apoptosis, combining FAM-FLICA® with 7-AAD can provide a more detailed picture of the overall health of the cell population by revealing the percentage of cells that are 7-AAD-negative (membrane intact live cells) and yet FAM-FLICA® positive (apoptotic) (Figures 3-6). FAM-FLICA® probes optimally excite at 488-492 nm with maximal emission at 515-535 nm. The vital staining dye 7-AAD optimally excites at 546 nm and emits at 647 nm. This significant difference in fluorescence emission wavelength between the green FAM (carboxyfluorescein) label on the FAM-FLICA® probe and the red 7-AAD vital dye simplifies flow cytometer gating and compensation. The FAM-FLICA® probe (apoptosis) is monitored on the FL-1 channel, while 7-AAD (necrosis) is monitored on FL-3. Combining the use of ICT’s FAM-FLICA® apoptosis detection probe with a membrane integrity dye like 7-AAD makes it easy to distinguish between necrosis and apoptosis within a single sample.