Annexin V-FITC 細胞凋亡檢測試劑盒

使用 Annexin V-FITC Apoptosis Assay Kit 檢測細胞凋亡和膜通透性。這種體外細胞凋亡測定採用綠色螢光膜聯蛋白 V-FITC 試劑來標記培養細胞樣品中暴露的磷脂酰絲氨酸。使用活/死染色劑碘化丙啶檢測膜受損細胞，這是晚期細胞凋亡或細胞壞死的標誌。通過流式細胞儀分析螢光訊號。

Background Annexin V is a member of a calcium and phospholipid binding family of proteins with vascular anticoagulant activity. Results from in vitro experiments indicate that it may play a role in the inhibition of blood coagulation by competing for phosphatidylserine (PS) binding sites with prothrombin. In healthy cells, PS is usually kept in the inner-leaflet (the cytosolic side) of the cell membrane. When a cell undergoes apoptosis, one of the earliest detectable indicators is the loss of membrane asymmetry. No longer restricted to the cytosolic part of the membrane, PS is translocated to the outer-leaflet and becomes exposed on the surface of the cell. With our Annexin V-FITC Apoptosis Assay Kit, ImmunoChemistry Technologies provides a proven method for quickly and easily distinguishing two populations of dying cells from viable cells using Annexin V-FITC and Propidium Iodide (PI) reagents. Cells in the early stages of apoptosis with intact cell membranes and surface -exposed PS will stain positive for Annexin V-FITC. PI is included in the apoptosis assay kit to identify late apoptotic and necrotic cells, which have lost plasma membrane integrity. These cells will become dually labeled with Annexin V-FITC and Propidium Iodide (green and red fluorescence). Live cells with intact plasma membranes will exclude PI and will remain unstained by the Annexin V-FITC probe, assuming no treatment or cell cycle-associated event temporarily exposes the normally internalized, negatively charged PS entity. The kit also includes a specially formulated, calcium-based binding buffer, which is required for Annexin V binding to occur.

Target Apoptosis, Exposed phosphatidyl serine

Excitation / Emission 488 nm / 530 nm

Method of Analysis Flow cytometry

Sample Type Cell culture

Storage 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls

2. Dilute 10X Binding Buffer 1:10 with diH2O.

3. Wash cells in ice-cold culture medium or PBS and resuspend in ice-cold 1X Binding Buffer.

4. Dilute Annexin V-FITC 1:10 in PBS.

5. Dilute Propidium Iodide (PI) 1:2 in PBS.

6. Add diluted Annexin V-FITC to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells).

7. Add diluted PI to each sample at a ratio of 1:20 (5 µL per 100 µL aliquot of cultured cells).

8. Incubate ~10 minutes, protected from light.

9. Dilute samples by adding 250 µL 1X Binding Buffer to each.

10. Analyze with a flow cytometer. Annexin V-FITC optimally excites at 494 nm and emits at 519 nm (typically read in FL1). PI optimally excites at 536 nm and has a peak emission at 617 nm (typically read in FL3).

Kit Contents

Annexin V-FITC, 200X, 50 µL, 1 vial, #6340 10X Binding Buffer, 1.7 mL, 3 vials, #6341 Propidium Iodide, 250 µg/mL, 1 mL, 1 vial, #638 Kit Manual