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  • : APT750
  • : 100 assays
  • Product Family Information

ApopNexin Annexin V FITC Apoptosis Kit

Description:
ApopNexin Annexin V FITC Apoptosis Kit
Trade Name:
  • ApopNexin
  • Chemicon (Millipore)
Qty/Pk:
100 assays
Product Overview:
INTRODUCTION

The ApopNexin™ FITC Apoptosis Detection Kit is designed to detect a specific biochemical change in the cell surface membrane that is considered to be a signature event of early apoptosis. These kits contain Annexin V conjugated with FITC, allowing for convenient quantitative assays. The counterstain, propidium iodide (PI), can be used to distinguish apoptotic cells with intact membranes from lysed, necrotic cells. These reagents must be used on live cells and are not applicable to previously sectioned, scraped, fixed or permeabilized cell samples. However, treatments that impact the cell membrane can be performed after the ApopNexin™ protocol.

Test Principle

Membrane Permeability Protocols

The counterstain, PI, is used to assay for the cell membrane permeability (lysis) in necrosis in Protocols #1, 2, and 3 below. Cell populations that potentially may be detected are as follows: Viable cells will be nonfluorescent; cells in the metabolically active stages of apoptosis will stain with ApopNexin™ but not with PI. Necrotic (lysed) cells with compromised membranes will bind both ApopNexin™ and PI. Late-stage apoptotic bodies may enter secondary necrosis if not deleted by phagocytosis, particularly in suspension cultures. As secondary necrotic (lysed) cells bind both ApopNexin™ and the DNA binding dye, staining with the dye indicates final necrosis but does not exclude cell death through apoptosis. Intermediate events are possible in some model systems. Thus, a working model of cell death kinetics should be considered to be very relevant to the design of an experimental system and to the interpretation of data.

ApopNexin™ Kits are qualified for use to detect apoptosis when directly applied to physically intact cell samples. They are not applicable to previously sectioned, scraped, fixed or permeabilized cell samples, but they may be used before such treatments. Refer to detailed Protocols #1-3 for these methods.
Background Information:
Annexins are a family of structurally related proteins that can bind specifically to cellular membranes (Cruetz, 1992). Annexin V has anticoagulant activity and is a monomer with a molecular weight of 35.8 kDa (Maurer-Fogy, 1989). It has a very high affinity for membranes containing the negatively charged phospholipid phosphatidylserine (PS). Specific binding is rapid and calcium-dependent. Its equilibrium dissociation constant is about 10-10 M in the presence of >1.5 mM Ca2+ (Andree, 1990), and specific binding is abolished if the Ca2+ is chelated by EDTA.

Binding of the ApopNexin™ conjugates to cell surface PS can be utilized for several kinds of rapid and convenient assays for apoptosis (Casciola-Rosen, 1996; England, 1996; Koopman, 1994; Martin, 1995; Vermes, 1995). Samples of suspended and adherent cells may be tested by flow cytometry or by microscopy using the protocols provided. Live, apoptotic or lysed/dead cells are separately detected by counterstaining with propidium iodide (PI).

Of all the aspects of apoptosis, the defining characteristic is a complete change in cellular morphology (Kerr, 1991). As may be conclusively examined by electron microscopy, the cell undergoes shrinkage, chromatin margination, membrane blebbing, nuclear condenstion and then segmentation, and division into apoptotic bodies that may be phagocytosed. There are many books in print (refer to Appendix: References) and review articles (e.g. Darzynkiewics, 1997

; Majno, 1995) describing all aspects of apoptosis.



Biological Mechanism

Phosphatidylserine (PS) is normally confined in the inner membrane leaflet of viable cells. The translocation of PS to the exposed membrane surface is an early event in apoptosis, where it serves as a signal for the attack of phagocytic cells. Translocation of PS is not due to a generalized change in the membrane but is controlled by the activity of a PS translocase enzyme (Martin, 1995). Another early membrane event that is characteristic of apoptosis is the eruption of cell surface blebs (Darzynkiewics, 1997). It is mainly at the surface blebs that the PS is exposed (Casciola-Rosen, 1996). Annexin V binding to apoptotic cells is a useful tool to quantitatively measure cells in the early and middle stages of apoptosis.

Necrosis, or cell death by metabolic arrest and lysis, as distinguished from apoptosis (Majno, 1995), is defined by a general swelling of the whole cell and its constituent organelles. Because this process is caused by early permeabilization of the cell membrane (lysis), necrosis is easily detected in vitro by exposure to a DNA binding dye such as PI. The exclusion of such a hydrophlic dye indicates cell viability. Cells in the metabolically active, early and middle stages of apoptosis do exclude the DNA binding dye. Only physically intact cells are amenable to this test (as opposed to chemically permeabilized or mechanically ruptured cells).



For Research Use Only; Not for use in diagnostic procedures
Applications:
The ApopNexin FITC Apoptosis Detection Kit for Flow Cytometry is designed to detect a specific biochemical change in the cell surface membrane that is considered to be a signature event of early apoptosis.
Key Applications:
Flow Cytometry
Species Reactivity:
Mammals
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Entrez Gene Summary:
The protein encoded by this gene belongs to the annexin family of calcium-dependent phospholipid binding proteins some of which have been implicated in membrane-related events along exocytotic and endocytotic pathways. Annexin 5 is a phospholipase A2 and protein kinase C inhibitory protein with calcium channel activity and a potential role in cellular signal transduction, inflammation, growth and differentiation. Annexin 5 has also been described as placental anticoagulant protein I, vascular anticoagulant-alpha, endonexin II, lipocortin V, placental protein 4 and anchorin CII. The gene spans 29 kb containing 13 exons, and encodes a single transcript of approximately 1.6 kb and a protein product with a molecular weight of about 35 kDa.
UniProt Summary:
FUNCTION: SwissProt: P08758 # This protein is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade.
SIZE: 320 amino acids; 35937 Da
SUBUNIT: Monomer. Binds ATRX (By similarity).
DOMAIN: SwissProt: P08758 A pair of annexin repeats may form one binding site for calcium and phospholipid.
SIMILARITY: Belongs to the annexin family. & Contains 4 annexin repeats.
Components:
  • Component / Part No. / Quantity / Storage
  • ApopNexin™ FITC / APT750a / 0.5 mL / 2-8°C
  • Binding Buffer, 4X / APT750b / 50 mL / 2-8°C
  • Propidium Iodide (PI), 20 μg/mL / APT750c / 1.6 mL / 2-8°C
Brand Family:
Chemicon
UniProt Number:
Storage Conditions:
ApopNexin™ FITC Apoptosis Detection Kit should be stored at 2-8°C and protected from light.

Precautions

· For research use only. Not for use in diagnostic procedures.

· Part no. APT750a contains 0.02% sodium azide.

· Part no. APT750c contains Propidium Iodide (PI), a suspected carcinogen. Handle with care using gloves and eye protection; do not ingest or contact to skin.
Detection Methods:
Fluorescent
Gene Symbol:
  • ANXA5
  • ANX5
  • Annexin-5
  • CBP-I
  • ENX2
  • PP4
  • PAP-I
Product Name:
ApopNexin Annexin V FITC Apoptosis Kit
Entrez Gene Number:
Materials Required but Not Delivered:
1. 5 mL culture tubes or 15 mL conical tubes

2. PBS; See Appendix: TECH NOTE #8

3. PBS containing 1 mg/mL BSA

4. Pipettes

5. RNase

6. Glass coverslips

7. Formalin, 37-38% (containing 10-15% methanol); See Appendix: TECH NOTE #4: Fixation

8. Methanol, 99+% spectrophotometry grade



Equipment Required

1. The instrumentation needed to collect bicolor data is either a flow cytometer with an excitation lam at 488 nm or a fluorescence microscope with a standard lamp. See Appendix: TECH NOTE #2: Fluorescence filters.

2. Desk-top centrifuge

Product Resources

View Gene Related Products for this Target