Annexin V- AF647/PI Apoptosis Detection Kit

Cat. No.: A1456538

GRADE & PURITY BioReagent, Ready-to-use, Biological Stain, for Microscopy, Suitable for Immunofluorescence(IF)

Overview

  Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner. 

  In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), PI. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative). This kit is suitable for the identification and enumeration of dead cells, such as apoptotic or necrotic cells, by flow cytometry.

Precautions  

1.     Please try to avoid light when using to slow down the quenching of fluorescence. 

2.     PI odide Solution is toxigenic and mutagenic; handle with care. 

3.     Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.   

Instruction for use  

1.     Dilute 10x Binding Buffer to 1x using distilled water (1 mL 10x Binding Buffer + 9 mL ddH2O). 

2.     Wash cells twice with cold PBS and then resuspend the desired amount of cells in Annexin V Binding Buffer at a concentration of 1.0-5.0 x 106 cells/mL. 

3.     Add 2 µl  Annexin V-AF647  and 2 µl PI to 100 µL of the cell suspension. 

4.     Add 100 µl of 1x Binding Buffer to each assay. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark. 

5.     Analyze by flow cytometry within 1 hr.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8°C long term (12 months). Store in the dark. Do not freeze.

Contents & Storage

A1456538	Components	20T	50T	100T	Storage	Quantity Per Test
A1456538A	10x Annexin V Binding Buffer	5 mL	10 mL	20 mL	2-8℃	200 μL / 0.5-1.0x10⁵ cells
A1456538B	Annexin V-AF647	40 μL	100 μL	200 μL	2-8℃. Store in the dark.	2 μL / 0.5-1.0x10⁵ cells
A1456538C	PI Staining Solution	40 μL	100 μL	200 μL	2-8℃. Store in the dark.	2 μL / 0.5-1.0x10⁵ cells

Note: The recommended number of cells to stain per test is 0.5-1.0x10⁵ cells.

Images

Flow Cytometry: Annexin V-AF647/PI Apoptosis Detection Kit (A1456538)

Annexin V-AF647/PI Apoptosis Detection Kit (A1456538) - Flow Cytometry

 Flow cytometric analysis was performed on Jurkat cells, either untreated (left panel) or treated with 2 µM camptothecin (C111281) for 16 hours (right panel), using the Annexin V-AF647/7-AAD Apoptosis Detection Kit (A1456538). The cells were stained with Annexin V-AF647 and PI and then analyzed. In the untreated sample (left panel), the majority of cells were negative for both Annexin V-AF647 and PI, indicating a viable, non-apoptotic population. Following the 16-hour treatment with camptothecin (right panel), three main cell populations were identified: 1. Viable, non-apoptotic cells: Annexin V-AF647 negative/PI negative. 2. Early apoptotic cells: Annexin V-AF647 positive/PI negative. 3. Late apoptotic and dead cells:  Annexin V-AF647 positive/PI positive.