廠牌: Immunochemistry 產地: 美國
Necrosis vs Apoptosis Assay Kit
Size: 50 - 100 tests
Background 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).
Reagent Name FAM-VAD-FMK, 7-AAD
Target Caspases, GC-rich DNA
Excitation / Emission FAM-FLICA – 492 nm / 529 nm 7-AAD – 546 nm / 647 nm
Method of Analysis Flow cytometry, Fluorescence microscope
Sample Type Cell culture
Country of Origin United States
Prepare samples and controls
Dilute Apoptosis Wash Buffer 1:10 with diH2O.
Reconstitute FAM-VAD-FMK 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 of diluted FLICA to 290 µL of sample).
Incubate approximately 1 hour.
Remove media, then wash cells: add 1X Apoptosis Wash Buffer and spin cells (twice).
Resuspend samples in 400 µL 1X Apoptosis Wash Buffer
Reconstitute 7-AAD with 260 µL DMSO
Add 7-AAD to each sample at 1:200 (e.g., add 2 µL to 400 µL of sample)
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