Annexin V-APC/DAPI Apoptosis Detection Kit - BioReagent, Biological Stain, for microscopy, Suitable for Immunofluorescence(IF), ready-to-use, high purity

Cat. No.: A1456534

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 4',6-diamidino-2-phenylindole （DAPI）, propidium iodide (PI), 7-AAD. 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.

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 0.5-1.0x106 cells /mL.

3.  Add 5 μL of Annexin V-APC and 2 μL of DAPI staining solution to 100 μL of cell suspension.

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

Precautions

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

2. DAPI 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.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 2-8℃ long term (12 months)

Contents & Storage

A1456534	Components	20 T	50 T	100 T	Storage	Quantity Per Test
A1456534A	10× Annexin V Binding Buffer	5 mL	10 mL	20 mL	2-8℃	200 μL per  0.5-1.0x105 cells
A1456534B	Annexin V-APC	100 μL	250 μL	500 μL	2-8℃. Store in the dark.	5 μL per  0.5-1.0x105 cells
A1456534C	DAPI Staining Solution	40 μL	100 μL	200 μL	2-8℃. Store in the dark.	2 μL per 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-APC/DAPI Apoptosis Detection Kit (A1456534)

Annexin V-APC/DAPI Apoptosis Detection Kit (A1456534) - 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-APC/DAPI Apoptosis Detection Kit (A1456534).
The cells were stained with Annexin V-APC and DAPI and then analyzed. In the untreated sample (left panel), the majority of cells were negative for both Annexin V-APC and DAPI, 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-APC negative/DAPI negative.
2. Early apoptotic cells: Annexin V-APC positive/DAPI negative.
3. Late apoptotic and dead cells: Annexin V-APC positive/DAPI positive.