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.
ICT’s Necrosis vs Apoptosis Assay kit simultaneously detects both apoptosis associated cytotoxicity events as well as cell death due to necrosis. Apoptotic cells are identified using ICT’s 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. Because 7-AAD alone may not detect cells in the early stages of apoptosis, it is essential to use it in combination with the green-fluorescent FAM-FLICA apoptosis detection reagent. Combining these two different types of fluorescent cell-status-indicator reagents within a single test can reveal a significant percentage of cells that are 7-AAD-negative (membrane intact live cells) and yet FAM-FLICA positive (apoptotic).
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.
Jurkat suspension cells were exposed to 1 µM of staurosporine for 4 hours at 37°C to induce apoptosis. Cells were dually stained with the green fluorescent FAM-FLICA poly caspase probe to detect apoptosis via caspase activity, and the red fluorescent vital dye 7-AAD to detect necrosis. The image shown in panel A reveals 4 populations of cells: 1) Live, unstained cells, which do not fluoresce. 2) Early stage apoptotic cells fluoresce green with FAM-FLICA. 3) Dually stained green and red fluorescing cells represent the population of Jurkat cells in mid-to-late stage apoptosis; these cells have active caspase enzymes and compromised cell membranes. 4) Necrotic cells fluoresce red. Panel B shows a corresponding differential interference contrast (DIC) image, which reveals cell morphology.
Flow cytometry analysis of Jurkat suspension cells to quantify four populations. (A) Cells were treated with a placebo (non-induced treatment with DMSO). (B) Cells were treated with 1 µM staurosporine for 4 hours to induce apoptosis via caspase activity. Cells were then dually stained with FAM-FLICA and 7-AAD, and analyzed using an Accuri C6 flow cytometer. FAM-FLICA was analyzed on FL-1 and 7-AAD was analyzed on FL-3. The key is shown in (C). Live, unstained cells do not fluoresce (lower left quadrant). Early apoptotic cells fluoresce green with FAM-FLICA. Dually stained green and red fluorescing cells represent the population of cells in mid to late apoptosis (these cells have active caspase enzymes and compromised cell membranes). Necrotic cells fluoresce red.
In the non-induced population (A), only 9.8% of cells were apoptotic (LR: 4.5% + UR: 6.3%), compared with 97.1% of the induced population (B; LR: 46.4% + UR: 50.7%).
• Target: FAM-VAD-FMK, 7-AAD
• Excitation/Emission: FAM-FLICA – 492 nm / 529 nm
7-AAD – 546 nm / 647 nm
• Method of Analysis: Fluorescence Microscope, Flow Cytometer
• Types of Samples: Cell culture
• Storage: 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping
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
Kit 9148: 100-200 tests
• FAM-FLICA Poly Caspase Reagent (FAM-VAD-FMK), 4 vials, #637
• 7-Aminoactinomycin D (7-AAD) vital dye, 2 0.26 mg vials, #6163
• 10X Apoptosis Wash Buffer, 2 60 mL bottles, #634
• Fixative, 6 mL, #636
• Kit Manual