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40295
Annexin V-PE Early Apoptosis Detection Kit
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Annexin V-PE Early Apoptosis Detection Kit #40295

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Flow cytometric analysis of Jurkat cells, untreated (left) or treated with camptothecin (10 uM, 4 hr; right), using Annexin V-PE Early Apoptosis Detection Kit.
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To Purchase # 40295
Cat. # Size Qty. Price
40295S		 1 Kit  (100 assays)

Custom Formulation

Product Includes Quantity (with Count)
Annexin V-PE Conjugate 1 x 200 µl
10X Annexin V Binding Buffer 2 x 1.5 ml
7-AAD Cell Staining Solution 47501 1 x 1 ml

Protocol

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Flow Cytometry, Annexin V-PE Conjugate, 7aad

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS) (#9808): To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Annexin-V Binding Buffer: To prepare 10ml 1X Annexin-V Binding Buffer: add 1ml 10X Binding Buffer to 9ml dH2O, mix.

B. Staining

  1. Collect cells of interest by centrifugation.
  2. Wash Cells with ice-cold PBS.
  3. Resuspend cells at 105 or 106 cells/ml with 1x Annexin V Binding Buffer. Aliquot 96μl of cell suspension in an assay tube.
  4. Add 2μl Annexin V-PE Conjugate and 10μl 7aad staining solution to each 96μl cell suspension. Allow cells to incubate 10 minutes on ice in the dark.
  5. Dilute the cell suspension to a final volume of 250μl/assay with ice-cold 1X Annexin V Binding Buffer. Analyze immediately.

posted May 2019

Protocol Id: 1824

Product Description

The Annexin V-PE Early Apoptosis Detection Kit enables researchers to identify early apoptotic cells within a cell population. Annexin V-PE conjugated protein binds to cell surfaces expressing phosphatidylserine, an early apotosis marker. Cells stained with 7-Aminoactinomycin D (7-AAD), a non-cell-permeable DNA dye, indicate necrotic cells. Cells stained with both 7-AAD and annexin V-PE demonstrate later stage apoptosis and early necrosis. This kit provides enough reagent to perform 100 assays, based on a 250 μl assay volume.

Specificity / Sensitivity

Species Reactivity:

All Species Expected

Background

Annexin V belongs to the annexin family of proteins consisting of over 160 members. All annexin family members share the same characteristic of Ca2+-dependent binding to negatively charged phospholipid surfaces (1). During early apoptosis, phosphatidylserine (PS) is translocated from the cytosolic side of the plasma membrane to the cellular surface. This translocation exposes PS to the extracellular environment with the plasma membrane left intact (2). Annexin V protein demonstrates high affinity, specificity, and sensitivity for PS and can be used as a marker of early apoptosis. In order to rule out "leaky" necrotic cells, annexin V must be used in tandem with reagents that determine the integrity of the cell membrane, such as propidium iodide (2).

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For Research Use Only. Not for Use in Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
This product is sold under license from Tau Technologies BV, U.S. Pat. 7,511,016 and foreign equivalents.
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