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Xpert Annexin V-FITC Apoptosis Detection Assay

The Xpert Annexin V-FITC Apoptosis Detection Assay offers a simple, fast and reliable method for the fluorescent detection of apoptotic cells and quantitative determination by flow cytometry. This assay is based on binding of FITC-labeled Annexin V to phosphatidylserine sites (PS) present on the membrane surface of apoptotic cells combined with the binding of propidium iodide (PI) to cellular DNA in necrotic cells in which cell membrane integrity has been completely compromised. This allows to distinguish between viable cells, early apoptotic cells and necrotic cells.

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Description

The Xpert Annexin V-FITC Apoptosis Detection Assay offers a simple, fast and reliable method for the fluorescent detection of apoptotic cells and quantitative determination by flow cytometry. This assay is based on binding of FITC-labeled Annexin V to phosphatidylserine sites (PS) present on the membrane surface of apoptotic cells combined with the binding of propidium iodide (PI) to cellular DNA in necrotic cells in which cell membrane integrity has been completely compromised. This allows to distinguish between viable cells, early apoptotic cells and necrotic cells.

Apoptosis is a gradual process leading to cell death, characterized by specific morphologic features. The translocation of PS from the inner to the outer leaflet of the plasma membrane, hence exposing PS to the external cellular environment, is one of the earliest phenomena of apoptosis, preceding loss of membrane integrity that occurs in later stages of apoptosis or during necrosis. Therefore when using a combination of green fluorescent FITC-labeled Annexin V (Annexin A5) and red fluorescent PI, viable cells are negative for both, whereas early apoptotic cells are Annexin V-positive, as Annexin V binds strongly to PS, even when labeled with FITC, but PI-negative (as intact membranes are impermeable to PI). Cells entering later stages of apoptosis or necrosis will be both Annexin V and PI-positive.

Albeit that this assay does not distinguish between cells that have died as a result of apoptosis and cells that have suffered another necrotic pathway, by measuring apoptosis over time, cells can often be followed from Annexin V-negative/PI-negative via Annexin V-positive/PI-negative to Annexin V-positive/PI-positive. Encountering all these three phenotypes at the same time within a cell population, or observing a shift through these three stages within a cell population over time, is thus a strong indicator of apoptosis.

Additional information

Quantidade

100 assays

Documents

Product Info – Xpert Annexin V-FITC Apoptosis Detection Assay

Protocols

Protocol

Prior to use:

  • Dilute 10x concentrated Binding Buffer with ultrapure water to 1x and keep on ice.
  • For the first washing step, keep some culture medium on ice.
  • For detection of adherent cells by Fluorescence Microscopy, please visit the product page of our website for procedure.
  • It is recommended to include a positive control for apoptosis.
  1. Harvest cells of interest by centrifugation. If working with adherent cells, first detach cells by trypsinizing or with Accutase®
  2. Gently wash cells with ice-cold culture medium
  3. Resuspend in 1x ice-cold Binding Buffer at a concentration of 105-106 cells/ml
  4. Transfer 500µl of the cell suspension to a 5-ml test tube
  5. Add 5µl of FITC-labeled Annexin V and 10µl of Propidium Iodide
  6. Mix gently and incubate for at room temperature (25ºC) in the dark for 15 min
  7. Gently wash cells with 1x ice-cold Binding Buffer and resuspend in 400µl of 1x Binding Buffer
  8. Analyze cell population by flow cytometry:

For FITC-labeled Annexin V binding to PS use the FITC signal detector (excitation λ=488nm, emission λ=530nm) (usually FL1)
For Propidium Iodide binding to DNA use the PE signal detector (excitation λ=535nm, emission λ=617nm) (usually FL2)

Notes

  • For detection by Fluorescence Microscopy, place stained cells on a glass slide and cover with a glass coverslip. Observe cells using a dual filter set for FITC and rhodamine, or alternatively with separate filters. Cells in early stage of apoptosis will demonstrate green staining on the plasma membrane, whereas cells at final stage of apoptosis or necrosis will show a red stain throughout the nuclei as well as a green stain on the plasma membrane.
  • For detection of adherent cells by Fluorescence Microscopy, please download separate protocol in the document section

Order Information

Reference Description Quantity
GTC21.0100 Xpert Annexin V-FITC Apoptosis Detection Assay 100 assays

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