壞死與細胞凋亡檢測試劑盒

壞死與細胞凋亡檢測試劑盒同時檢測細胞凋亡相關的細胞毒性事件以及壞死引起的細胞死亡。這種簡單直接的工具使研究人員能夠了解其細胞群的整體健康狀況。此外，該試劑盒對研究他們的新藥或治療劑的效果的研究人員很有用，因為它允許他們評估他們的實驗結果並評估細胞的整體治療。使用螢光顯微鏡或流式細胞儀分析螢光信號。

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

Storage 2-8°C

Country of Origin United States

Protocols

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 Contents

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