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CBA059 Annexin V-FITC Apoptosis Detection Kit II

CBA059
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Overview

Replacement Information

Key Specifications Table

Detection Methods
Fluorescence

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      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.
      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