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CBA059 Annexin V-FITC Apoptosis Detection Kit II

CBA059
Purchase on Sigma-Aldrich

Overview

Replacement Information

Key Spec Table

Detection Methods
Fluorescence

Products

Catalogue NumberPackaging Qty/Pack
CBA059-1KIT Glass bottle 1 kit
Description
OverviewA convenient kit for the identification of changes in the plasma membrane that occur during apoptosis. In apoptotic cells, phosphoatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, allowing for the detection of PS on the cell surface. In the presence of Ca 2+, Annexin V exhibits a high affinity for PS and binds cells with exposed PS. Annexin V, when conjugated to a fluorophore, can be used to monitor apoptopic cells using flow cytometry or immunofluorescence microscopy.
Catalogue NumberCBA059
Brand Family Calbiochem®
Materials Required but Not Delivered 1X PBS
Flow cytometer or fluorescence microscope
References
ReferencesDarzynkiewicz, Z., et al. 1997. Cytometry 27, 1.
Frey, T. 1997. Cytometry 28, 253;
Boersma, A.W.M., et al. 1996. Cytometry 24, 123.
White, E. 1996. Genes and Devel. 10, 1.
Martin, S.J., et al. 1995. J. Exp. Med. 182, 1545.
Koopman, G., et al. 1994. Blood 84, 1415.
Wyllie, A.H.,1993. Br. J. Cancer 67, 205.
Darzynkiewicz, Z., et al. 1992. Cytometry 13, 795.
Fadok, V.A., et al. 1992. J. Immunology 148, 2207.
Schmid, I., et al. 1992. Cytometry 13, 204.
Kerr, J.F.R., et al. 1972. Cancer 26, 239.
Product Information
Detection methodFluorescence
Form20 Tests
FormatFlow cytometry or fluorescence microscopy
Kit containsAnnexin V-FITC, Binding Buffer, Propidium Iodide and a user protocol.
Quality LevelMQ100
Applications
Key Applications Flow Cytometry
Immunofluorescence
Biological Information
Assay time30 min
Sample TypeIntact cells
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Intended useThe Calbiochem® Annexin V-FITC Apoptosis Detection Kit II is a non-isotopic system that can be used to detect phosphatidylserine on the outer leaflet of the cell membrane of apoptotic cells using flow cytometry or fluorescence microscopy.
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Storage +2°C to +8°C
Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Kit containsAnnexin V-FITC, Binding Buffer, Propidium Iodide and a user protocol.
Specifications

Documentation

Annexin V-FITC Apoptosis Detection Kit II SDS

Title

Safety Data Sheet (SDS) 

Annexin V-FITC Apoptosis Detection Kit II Certificates of Analysis

TitleLot Number
CBA059

References

Reference overview
Darzynkiewicz, Z., et al. 1997. Cytometry 27, 1.
Frey, T. 1997. Cytometry 28, 253;
Boersma, A.W.M., et al. 1996. Cytometry 24, 123.
White, E. 1996. Genes and Devel. 10, 1.
Martin, S.J., et al. 1995. J. Exp. Med. 182, 1545.
Koopman, G., et al. 1994. Blood 84, 1415.
Wyllie, A.H.,1993. Br. J. Cancer 67, 205.
Darzynkiewicz, Z., et al. 1992. Cytometry 13, 795.
Fadok, V.A., et al. 1992. J. Immunology 148, 2207.
Schmid, I., et al. 1992. Cytometry 13, 204.
Kerr, J.F.R., et al. 1972. Cancer 26, 239.

Brochure

Title
Kit SourceBook - 2nd Edition EURO
User Protocol

Revision29-August-2019 JSW
Form20 Tests
FormatFlow cytometry or fluorescence microscopy
Detection methodFluorescence
StorageUpon arrival store the entire contents of the kit at 4°C.
Intended useThe Calbiochem® Annexin V-FITC Apoptosis Detection Kit II is a non-isotopic system that can be used to detect phosphatidylserine on the outer leaflet of the cell membrane of apoptotic cells using flow cytometry or fluorescence microscopy.
BackgroundApoptosis is a fundamental mode of cell death that serves as an important regulatory function during normal development, in tissue homeostasis, and in some disease processes. In normal viable cells phosphatidylserine (PS) is located on the cytoplasmic surface of the cell membrane. Upon induction of apoptosis, rapid alterations in the organization of phospholipids in most cell types occurs leading to exposure of PS on the cell surface. Recognition of PS by phagocytes in vivo results in the removal of cells programmed to die, thus apoptosis is not commonly associated with the local inflammatory response that accompanies necrosis. In vitro detection of externalized PS can be achieved through interaction with the anticoagulant annexin V. In the presence of calcium, rapid high affinity binding of annexin V to PS occurs. PS translocation to the cell surface precedes nuclear breakdown, DNA fragmentation, and the appearance of most apoptosis-associated molecules making annexin V binding a marker of early-stage apoptosis.
Principles of the assay

Figure 1: Annexin/Phosphatidyl Serine in Early Stages Apoptosis


The Calbiochem® Annexin V-FITC Apoptosis Detection Kit II utilizes annexin V conjugated to FITC to bind to phosphatidylserine on the outer surface of cells undergoing apoptosis. Fluorescence is then detected by flow cytometry or fluorescence microscopy. Since membrane permeabilization is also observed in necrosis, necrotic cells will also bind Annexin V-FITC. Propidium iodide is included to help distinguish between viable, early apoptotic, and necrotic or late apoptotic cells. Necrotic cells will bind Annexin V-FITC and stain with propidium iodide while propidium iodide will be excluded from viable (FITC negative) and early apoptotic (FITC positive) cells. In the absence of phagocytosis final stages of apoptosis involve necrotic-like disintegration of the total cell, thus cells in late apoptosis will be labeled with both FITC and propidium iodide.
Materials providedThere are sufficient reagents to label 20 samples with annexin V-biotin.

• Annexin V-FITC (Kit Component No. JA9201-100UL): 1 vial, 100 µl, recombinant, human annexin V, (expressed in E. coli, MW 35,800, >98% pure by SDS-PAGE and HPLC) conjugated to FITC and supplied in 100 mM NaCl, 50 mM Tris-HCl, 1% BSA, 0.02% NaN₃, pH 7.4
• Binding Buffer (Kit Component No. JA9202-50ML): 1 vial, 50 ml, supplied as a 4X stock solution
• Propidium Iodide (Kit Component No. JA9203-EA): 1 vial, supplied at 20 µg/ml
Materials Required but not provided 1X PBS
Flow cytometer or fluorescence microscope
Reagent preparation• 1X Binding Buffer: Dilute the Binding Buffer 1:4 with dH2O (e.g., 50 ml Binding Buffer + 150 ml dH2O). This will yield a working concentration of 140 mM NaCl, 10 mM HEPES/NaOH, 2.5 mM CaCl2.
Detailed protocol1. Wash cells with 1X PBS and centrifuge at 1000 rpm to pellet the cells. Discard the supernatant and repeat for a total of 2-3 washes.
2. Resuspend the cell pellet in 1X Binding Buffer to a final concentration of 2-5 X 105 cells/ml.
3. Transfer 195 µl cell suspension to a clean tube and add 5 µl Annexin V-FITC. Mix and incubate for 10 min at room temperature.
4. Wash the cells with 1X Binding Buffer and centrifuge at 1000 rpm to pellet the cells. Discard the supernatant.
5. Resuspend the cell pellet in 190 µl 1X Binding Buffer and add 10 µl Propidium Iodide (final concentration = 1 µg/ml).
6. Analyze by flow cytometry or fluorescence microscopy