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PF036 Annexin V-Biotin Apoptosis Detection Kit

Annexin V-Biotin Apoptosis Detection Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
PF036
  
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Fluorescence
      Description
      OverviewNovel RAPID™ protocol and easy sample preparation. Optimized for flow cytometry and fluorescence microscopy applications. Useful for detection of membrane changes during apoptosis. Also useful in conjugation with GFP or other fluorophores.
      User provides appropriately labeled streptavidin.
      Catalogue NumberPF036
      Brand Family Calbiochem®
      Materials Required but Not Delivered• Streptavidin or anti-biotin conjugated to a fluorescent molecule such as fluorescein isothiocyanate (FITC), cyanine 3 (Cy3), Alexa™488, or phycoerythrin (PE)
      • PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM KH2PO4)
      • 2-20 µl, 20-200 µml, and 200-1000 µl precision pipettors with disposable tips
      • Microcentrifuge tubes
      • Adjustable speed microcentrifuge
      • Glass microscope slides (for microscope analysis only)
      • Glass coverslips (for microscope analysis only)
      • dH2O
      • Ice
      • Fluorescence microscope or flow cytometer
      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
      DeclarationSold under license of U.S. Patent 5,834,196.
      Form100 Tests
      FormatFlow cytometry or fluorescence microscopy
      Kit containsAnnexin V-biotin, 5X Binding Buffer, Propidium Iodide, RAPID™ Media Binding Reagent, and a user protocol.
      Positive controlApoptotic Jurkat cells (dexamethasone or anti-Fas treated)
      Applications
      Key Applications Flow Cytometry
      Immunofluorescence
      Biological Information
      Assay time15 - 30 min
      Sample TypeIntact cells
      Species Reactivity
      • A Broad Range Of Species
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Annexin V-Biotin Apoptosis Detection Kit is a non-isotopic system for the identification of cell membrane alterations that accompany programmed cell death. Detection may be performed by flow cytometry or by fluorescence microscopy using various fluorescent streptavidin or anti-biotin conjugates.
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsAnnexin V-BIOTIN kit components are shipped on gel ice pack. Upon receipt, store kit at 4°C. To avoid reagent loss in tube caps, briefly pulse spin all small tubes before removing caps. See Precautions and Recommendations.
      Do not freeze Yes
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsAnnexin V-biotin, 5X Binding Buffer, Propidium Iodide, RAPID™ Media Binding Reagent, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalog Number GTIN
      PF036 0

      Documentation

      Annexin V-Biotin Apoptosis Detection Kit Certificates of Analysis

      TitleLot Number
      PF036

      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

      Revision28-October-2010 RFH
      Form100 Tests
      FormatFlow cytometry or fluorescence microscopy
      Detection methodFluorescence
      Speciesa broad range of species
      StorageAnnexin V-BIOTIN kit components are shipped on gel ice pack. Upon receipt, store kit at 4°C. To avoid reagent loss in tube caps, briefly pulse spin all small tubes before removing caps. See Precautions and Recommendations.
      Intended useThe Calbiochem® Annexin V-Biotin Apoptosis Detection Kit is a non-isotopic system for the identification of cell membrane alterations that accompany programmed cell death. Detection may be performed by flow cytometry or by fluorescence microscopy using various fluorescent streptavidin or anti-biotin conjugates.
      BackgroundApoptosis is a fundamental mode of cell death that performs a 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
      In this assay biotinylated Annexin V is used with fluorescent conjugates of streptavidin or anti-biotin allowing detection of apoptosis by flow cytometry or by fluorescence microscopy. Since membrane permeabilization is observed in necrosis, necrotic cells will also bind Annexin V-BIOTIN. Propidium iodide is used to distinguish between viable, early apoptotic, and necrotic or late apoptotic cells. Necrotic cells will bind Annexin V-BIOTIN and stain with propidium iodide while propidium iodide will be excluded from viable (fluorescence negative) and early apoptotic (fluorescence 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 fluorescence and propidium iodide.

      A RAPID protocol has been developed for Annexin V-Biotin binding directly in tissue culture media. This obviates the need for tedious centrifugation and wash steps that increase the occurrence of mechanical membrane disruption. In addition, since apoptosis is a dynamic process that is ongoing once cells are removed from culture conditions and continues throughout experimental processing, the RAPID protocol is recommended for the detection of cells in early apoptosis.
      Materials providedNote: The Annexin V-BIOTIN Apoptosis Detection Kit supplies sufficient reagents for 100 tests.
      • ANNEXIN V-BIOTIN (Kit Component No. JA1252-125UL): 200 mg/ml recombinant Annexin V conjugated to biotin containing 1% BSA and 0.02% sodium azide
      • 5X BINDING BUFFER (Kit Component No. JA1652-12.5ML): Supplied as a 5-fold concentrated stock containing 20% BSA
      • MEDIA BINDING REAGENT (Kit Component No. JA1653-1.2ML): A reagent designed to enhance binding of Annexin V to PS in tissue culture media
      • PROPIDIUM IODIDE (Kit Component No. JA1654-1.5ML): supplied as 30 mg/ml propidium iodide in PBS
      Materials Required but not provided• Streptavidin or anti-biotin conjugated to a fluorescent molecule such as fluorescein isothiocyanate (FITC), cyanine 3 (Cy3), Alexa™488, or phycoerythrin (PE)
      • PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4-7H2O, 1.4 mM KH2PO4)
      • 2-20 µl, 20-200 µml, and 200-1000 µl precision pipettors with disposable tips
      • Microcentrifuge tubes
      • Adjustable speed microcentrifuge
      • Glass microscope slides (for microscope analysis only)
      • Glass coverslips (for microscope analysis only)
      • dH2O
      • Ice
      • Fluorescence microscope or flow cytometer
      Precautions and recommendations• For optimal results READ THESE INSTRUCTIONS BEFORE USING THIS KIT.
      • To avoid reagent loss in tube caps, briefly pulse spin all tubes before removing caps.
      • Propidium iodide may be harmful by ingestion or absorption through skin and may cause irritation to the eyes. It should be treated as a possible mutagen.
      • Gloves, lab coat, and protective eyewear should be worn.
      • Apoptosis detection with the Annexin V-Biotin kit should be performed on live cells. Fixation procedures may interfere with the observation of the fluorescent signal. It may be possible to fix cells following labeling, however excess calcium should be provided to prevent the reversal of the Annexin-PS interaction.
      • Analysis should be performed immediately after binding of Annexin V-Biotin since apoptosis will be an ongoing process. Loss of fluorescent signals may occur over time.
      • Propidium iodide and Annexin V-Biotin is light sensitive. Minimize exposure to light. Use a covered ice bucket or aluminum foil where necessary.
      • Successful detection of early apoptosis with Annexin V-Biotin is dependent upon several factors including cell type and level of membrane PS, amount of PS that is exposed during apoptosis, method (or agent) inducing apoptosis, time of apoptotic induction, as well as degree of mechanical manipulation. It may be necessary to optimize these procedures for your particular application.
      • When using streptavidin or anti-biotin conjugated to a fluorescent molecule such as phycoerythrin or cyanine 3 with emission wavelengths that overlap those of propidium iodide it is suggested that 1 µg/ml 7-aminoactinomycin D (7-AAD) be used in place of propidium iodide. 7-AAD can be excited by the argon-ion laser and emits beyond these wavelengths.
      Detailed protocolThree protocols are provided. One for the binding of Annexin V-Biotin directly in tissue culture media (RAPID Annexin V Binding), one for the binding of Annexin V-Biotin in binding buffer (Conventional Annexin V Binding) following a PBS wash, and another for binding of Annexin V-Biotin with adherent cells. The RAPID protocol is recommended where possible.

      For all protocols prepare enough 1X Binding Buffer (0.5 ml/sample for RAPID binding and 1.0 ml/sample for Conventional binding) by diluting the 5X Binding Buffer concentrate 1:5 with dH2O. Place on ice.

      RAPID Annexin V-Biotin Binding

      1. Adjust the cell suspension concentration to ~1 x 106 cells/ml.
      2. Transfer 0.5 ml of cell suspension from tissue culture flask (5 x 105 cells) to a microfuge tube.
      3. Add 10 µml Media Binding Reagent.
      4. Add 1.25 µl Annexin V-Biotin.
      5. Incubate 15 min at room temperature (18-24°C) in the dark.
      6. Centrifuge at 1000 x g for 5 min at room temperature. Remove media.
      7. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer.
      8. Add 15 µl of fluorescent streptavidin conjugate (diluted to 15 µg/ml in 1X Binding Buffer) or 15 µl of fluorescent anti-biotin conjugate (diluted to 10-20 µg/ml in 1X Binding Buffer).
      9. Add 10 µl Propidium Iodide (or 1 µg/ml 7-AAD if a Cy3 or PE conjugate is used, see Precautions and Recommendations).
      10. Place samples on ice and away from light.
      11. Analyze by flow cytometry or fluorescence microscopy immediately.

      Conventional Annexin V-Biotin Binding

      1. Adjust the cell suspension concentration to ~1 x 106 cells/ml.
      2. Transfer 0.5 ml of cell suspension from tissue culture flask (5 x 105 cells) to a microfuge tube.
      3. Centrifuge at 1000 x g for 5 min at room temperature. Remove media.
      4. Gently resuspend cells in 0.5 ml cold PBS.
      5. Centrifuge at 1000 x g for 5 min at room temperature. Remove PBS.
      6. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer.
      7. Add 1.25 µl Annexin V-BIOTIN.
      8. Incubate 15 min at room temperature (18-24°C) in the dark.
      9. Centrifuge at 1000 x g for 5 min at room temperature. Remove supernatant.
      10. Gently resuspend cells in 0.5 ml cold 1X Binding Buffer.
      11. Add 15 µl of fluorescent-streptavidin conjugate (diluted to 15 µg/ml in 1X Binding Buffer) or 15 µl of fluorescent anti-biotin conjugate (diluted to 10-20 µg/ml in 1X Binding Buffer).
      12. Add 10 µl Propidium Iodide (or 1 µg/ml 7-AAD if a Cy3 or PE conjugate is used, see Precautions and Recommendations).
      13. Place samples on ice and away from light.
      14. Analyze by flow cytometry or fluorescence microscopy immediately.

      Annexin V-Biotin Binding with Adherent Cells

      1. Transfer media from flask of adherent cells to a 15 ml conical tube and place on ice.
      Note: This media will contain cells that have become detached from the flask during
      the cell death process.
      2. Gently wash cells in flask with 10 ml PBS. Remove PBS.
      3. Add 1-2 ml 0.5X trypsin and incubate just until cells appear detached by microscopic evaluation.
      4. Release cells from flask with firm tapping.
      5. Gently resuspend cells in media from step 1 containing non-trypsinized detached (floating) cells OR 1X cold binding buffer to approximately 1 x 106 cells/ml.
      6. Transfer 0.5 ml of cell suspension to a microfuge tube and continue as described in the RAPID Protocol steps 3-11 OR the Conventional Annexin V Binding Protocol steps 7-14.
      Assay characteristics and examplesAnalysis by Flow Cytometry {using Streptavidin (SA)-FITC as an example}

      A flow cytometer emitting an excitation light at 488 nm from an argon ion laser should be used to quantify the Annexin V-Biotin-SA-FITC and propidium iodide signals. To set up the flow cytometer use apoptosis-induced cells stained with Annexin V-Biotin-SA-FITC only and apoptosis-induced cells labeled with only propidium iodide. The FITC signal can be detected by FL1 (FITC detector) at 518 nm. Propidium iodide fluoresces at 620 nm and can be detected by FL2 (the phycoerythrin fluorescence detector). Perform necessary adjustments to minimize overlap between these two measurements. The log of FITC fluorescence should be displayed on the X axis and the log of propidium iodide fluorescence; on the Y axis of the data report. The flow cytometer may also be programmed to display additional parameters such as forward and side (90°) scatter.

      Analysis by Fluorescence Microscopy

      Transfer 25-50 µl of cell suspension following addition of propidium iodide onto a glass microscope slide. Cover with a glass coverslip and view immediately using a fluorescence microscope equipped with FITC and rhodamine filter sets. Addition of mounting media to the sample will dilute the calcium concentration and may result in the reversal of Annexin V-Biotin-SA-FITC binding.

      Evaluation of results

      Figure 1: Principle of the Assay


      Alterations in light scattering properties as measured by flow cytometry can also be used to monitor cells undergoing apoptosis. For example, untreated Jurkat cells have high forward scatter. Upon induction of apoptosis Jurkat cell shrinkage and membrane blebbing occurs and a marked increase in side scatter is observed. A combination of changes in light scatter and Annexin V-Biotin binding may be used to confirm or support conclusions drawn from measurements of a single parameter.

      Fluorescence Microscopy

      Using the FITC filter (blue light) on a fluorescence microscope positive Annexin V-Biotin-SA-FITC staining will appear bright apple green on the cell membrane surface. Using the rhodamine filter (green light) cells that are positive for propidium iodide will appear with various intensities of yellow-red throughout the cytoplasm.

      Early apoptotic cells will be unstained with propidium iodide or show background fluorescence, while necrotic and late apoptotic cells will have yellow-red cytoplasm and red nuclear staining with green fluorescence surrounding the perimeter of the cell. Membrane blebbing and cell shrinkage may be observable with late apoptotic cells.

      Figure 2: Flow Cytometry

      A sample cytogram is shown above. Viable cells do not bind Annexin V-Biotin or propidium iodide as reflected in the lower left-hand quadrant of the dot plot. Early apoptotic cells with exposed PS but intact cell membranes bind Annexin V-Biotin but exclude propidium iodide. Fluorescence from this population is reported in the lower right-hand quadrant. Necrotic or apoptotic cells in terminal stages will be both Annexin V-Biotin and propidium iodide positive and will be reported in the upper right-hand quadrant. A small percentage of normal cell death should be expected in routine cultures of untreated cells.


      Figure 3: Validation

      RAPID Annexin V-Biotin binding is identical to results with the CONVENTIONAL protocol. Jurkat cells were incubated with 0.5 mg/ml actinomycin D for eieither 5 (Middle) or 19 (Bottom) h. Untreated Jurkat cells are shown in the Top panel of each section. Annexin V-Biotin was then bound in either binding buffer (using the Conventional Protocol) or directly in media (RAPID Protocol). SA-FITC was used to detect bound Annexin V-Biotin.


      Figure 4: Detection of Annexin V-BIOTIN binding with Fluorescein and Alexa™488 conju

      Jurkat cells were incubated with 0.5 µg/ml actinomycin D for either 5 (Middle) or 19 (Bottom) h. Untreated Jurkat are shown in the Top panel of each section. Annexin V-Biotin was then bound directly in media (RAPID protocol). SA-FITC, SA-Alexa™488 or anti-Biotin-Alexa™488 was used to detect bound Annexin V-Biotin. Propidium iodide was added to distinguish between Annexin V positive apoptotic and necrotic cells.


      Figure 5: Detection of Annexin V-Biotin binding with cyanine 3 (Cy3) and phycoerythrin (PE

      Jurkat cells were incubated with 0.5 µg/ml actinomycin D for either 5 (Middle) or 19 (Bottom) h. Untreated Jurkat are shown in the Top panel of each section. Annexin V-Biotin was then bound directly in media (RAPID protocol). SA-Cy3 or SA-PE was used to detect bound Annexin V-Biotin. To avoid spectral overlap between Cy3, PE, and propidium iodide, 7-aminoactinomycin D (7AAD) was added for discrimination of necrotic cells from Annexin V-Biotin positive apoptotic cells.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.
      Alexa™488 is a trademark of Molecular Probes, Inc.