Annexin V Binding

Guava Nexin Assay
for detection of early apoptosis

The Guava Nexin® Annexin V Assay offers a simple, reproducible, and reliable assessment of early apoptosis. The assay relies on the translocation of phosphatidyl serine (PS) to the outer surface of the cell membrane, an event often associated with the onset of apoptosis. 

Simple, sensitive, and reproducible 

A mix-and-read assay for monitoring externalization of PS through the binding of Annexin V to the exposed PS, the automated single-cell analysis assay is the choice for monitoring apoptosis due to its sensitivity and reproducibility. Annexin V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine (PS), a membrane component normally localized to the internal face of the cell membrane. Early in the apoptotic pathway, molecules of PS are translocated to the outer surface of the cell membrane where Annexin V can readily bind to them.

Two-dye strategy

The assay relies on a two-dye stategy: 1) Annexin V-PE to detect PS on the external membrane of apoptotic cells and 2) 7-AAD, a cell impermeant dye, as an indicator of membrane structural integrity. 7-AAD is excluded from live, healthy cells and early apoptotic cells, but permeates late-stage apoptotic and dead cells. The two-dye strategy allows for identification of four populations:

• Non-apoptotic cells: Annexin V (–) and 7-AAD (–)
• Early apoptotic cells: Annexin V (+) and 7-AAD (–)
• Late stage apoptotic and dead cells: Annexin V (+) and 7-AAD (+)
• Mostly nuclear debris: Annexin V (–) and 7-AAD (+)

Key features

• Mix-and-read, tube- or 96-well plate protocol
• Analysis of apoptosis in single cells
  
• Robust and reproducible: Z’ factor > 0.7
• Works with adherent and suspended cells
• Staining and data acquisition in ≤1 hours
• Provides direct, absolute cell counts as well as population percentage
• Data compatible with third party analysis software
• Runs on Guava PCA, PCA-96, EasyCyte Mini, and EasyCyte Plus Systems

Key references

Dumont, P, Leu, JI, Della Pietra, AC, George, DL, Murphy, M. The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nature Genetics. 2003;33(3):357-365.

Pedersen IM, Kitada S, Leoni LM, Zapata JM, Karras JG, Tsukada N, Kipps TJ, Choi YS, Bennett F, Reed JC. Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1. Blood. 2002;100(5):1795-1801.

Wu Q, Kirschmeier P, Hockenberry T, Yang TY, Brassard DL, Wang L, McClanahan T, Black S, Rizzi G, Musco ML, Mirza A, Liu SX. Transcriptional Regulation during p21(WAF1/CIP1)-induced Apoptosis in Human Ovarian Cancer Cells. J Biol Chem. 2002;277(39):36329–36337.

van den Eijnde SM, et al. In situ detection of apoptosis during embryogenesis with Annexin V: From whole mount to ultrastructure. Cytometry. 1997;29:313-320.

Majno G, Joris I. Apoptosis, oncosis, and necrosis: An overview of cell death. Am J

Vermes I, et al. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Meth. 1995;184:39-51.

Wyllie AH, Apoptosis. Br J Cancer. 1993; 67:205-208.

Schmidt I, et al. Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry.1992;13:204-208.

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