pSIVA™ Microscopy Kit

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1x105 HeLa cells were seeded on Millicell® EZ Slide 4-well glass slides (sterile) and cultured in 0.5 ml RPMI medium supplemented with 2 mM CaCl2. Cells were treated with 1 µM of the apoptosis inducing agent staurosporine (Figures E, F, G, H) or left untreated (Figures A, B, C, D). pSIVA–IANBD + PI were added to the media according to kit instructions. Cells were imaged post treatment with the Zoe™ Fluorescent Cell Imager at 0 minutes, 60 minutes, 120 minutes and 270 minutes. Images A-H are merged images of the red and green fluorescence channels. Early apoptotic cells were stained with pSIVA-IANBD and are shown in green while late apoptotic/dead cells were stained with PI and are shown in red.

Millicell® is a registered trademark of Merck KGaA, Darmstadt, Germany

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  • pSIVA™ REAL-TIME Apoptosis Fluorescent Microscopy Kit
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  • Product Type
    Kits
1 Formats Available
    Product CodeApplicationsDatasheetMSDSPack SizeList PriceQuantity
    APO004IC, IF, LIdatasheet pdfdatasheet pdf datasheet pdf1 Kit
    APO004
    Summary
    Secondary Antibodies
    Negative Isotype Controls
    Useful Reagents
    Positive Controls
    Histology Controls
    More Images
    References
    Reviews
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    • The process of apoptosis is undertaken in several stages defined by specific cellular morphologies. One of the earlier stages of apoptosis is a change of the plasma membrane’s phospholipid asymmetry. This rearrangement results in the translocation of phosphatidylserine (PS) from the inner to the outer plasma membrane (in non-apoptotic cells PS is exclusively located to the inner plasma membrane). However, apoptosis is reversible until reaching a certain point in the pathway and until then PS exposure can be considered as a transient event. The event defining whether the cell can be rescued and continues living is the onset of mitochondrial outer membrane permeabilization (MOMP) (Chipuk et al. 2006). Prior to reaching this point, PS exposure may be transient as molecules can relocate back to the inner plasma membrane (a phenomenon known as “PS flipping”) (van der Mark et al. 2013).

      The pSIVA™ (polarity-Sensitive Indicator of Viability & Apoptosis) probe is a biosensor conjugated to the green emitting IANBD dye (excitation maximum 488 nm, emission maximum 530 nm) and only fluoresces when bound to PS in the presence of Ca2+ (Kim et al. 2010a, 2010b). The method thereby allows the analysis of kinetic apoptosis events in real time by live cell imaging and immunofluorescence/immunocytochemistry. In contrast to other PS detection based assays (e.g. annexin V) the pSIVATM Real-Time Apoptosis Fluorescent Microscopy Kit does not require washing steps as you can simply add the probe and start analyzing.
    • Intended Use
    • Product Form
    • Reconstitution
    • Preparation
    • Preservative Stabilisers
    • Purity
    • Approx. Protein Concentrations
    • Reagents In The Kit
      pSIVA-IANBD 200 μl
      Propidium Iodide Staining Solution 500 μl
    • Preparing The Antibody
    • Test Principle
    • Buffer Solution
    • Storage
      Store at +4oC. DO NOT FREEZE.
      This product should be stored undiluted. This product is photosensitive and should be protected from light.
    • Shelf Life
      6 months from date of despatch
    • Acknowledgements
      pSIVATM is a trademark of Novus Biologicals and is protected under patent no. 8.541.549.
    • Regulatory
      For research purposes only
    • This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.

    • Application NameYesNoMin DilutionMax Dilution
      ImmunocytochemistryRefer to Instructions For Use
      ImmunofluorescenceRefer to Instructions For Use
      Live Cell ImagingRefer to Instructions For Use

    • Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using appropriate negative/positive controls.
    • Technical Advice
    • Recommended Protocol
    • ELISA
    • Immunohistology
    • Histology Positive Control Tissue
    • Immunofluorescence
    • Western Blotting
    • Instructions For Use
      Prior to commencing the microscopy experiment, please ensure that your cell culture medium contains between 1-2 mM Ca2+. Ca2+ is essential for binding of the pSIVA-IANBD probe to exposed phosphatidylserine (Kim et al. 2010b). If Ca2+ levels are insufficient, supplement the culture medium with 2 mM Ca2+.

      1. Seed cells into culture plates and allow cells to adhere.
      2. Optional. After 24 hours exchange the culture medium for medium containing 2 mM Ca2+, if required.
      3. Optional. Induce apoptosis by treating cells with apoptosis inducing agents such as staurosporine or camptothecin.
      4. Add 10–20 µl/ml* of the pSIVA-IANBD probe to cells. Mix gently by moving culture plates
      backwards and forwards and side to side to ensure even distribution of the probe. DO NOT PIPETTE TO MIX.
      5. Optional. If distinction between apoptotic and necrotic/dead cells is desired, add between 5–10 µl/ml* of propidium iodide (PI) to cells. Mix gently by moving plates backwards and forwards and side to side to ensure even distribution of PI. DO NOT PIPETTE TO MIX.
      6. Observe cells under microscope using the green fluorescence filter for pSIVA-IANBD (excitation maximum 488 nm, emission maximum 530 nm) and the red fluorescence filter for PI (excitation maximum 535 nm, emission maximum 617 nm) visualization.
      * The stated pSIVA-IANBD and PI quantities are guidelines only and may have to be optimized.

      Instructions for use can be found at www.bio-rad-antibodies.com/uploads/IFU/APO004.pdf

    Additional pSIVA™ Microscopy Kit Formats

    Formats Applications Sizes available
    pSIVA™ Microscopy Kit : IANBD (Green Fluorescence) IC, IF, LI 1 Kit
    • Copyright © 2016 Bio-Rad

    Recommended Secondary Antibody

      Recommended Negative Isotype Control

        Useful Reagents

          DescriptionProduct CodePack SizeApplicationsList PriceQuantity
          Annexin V:APC Assay KitANNEX200APC200 TestsF
          ANNEX200APC
          Annexin V:APC Assay KitANNEX50APC50 TestsF
          ANNEX50APC
          Annexin V:Biotin Assay KitANNEX100B100 TestsF
          ANNEX100B
          Annexin V:Biotin Assay KitANNEX20B20 TestsF
          ANNEX20B
          Annexin V:Biotin Assay KitANNEX300B300 TestsF
          ANNEX300B
          Annexin V:FITC Assay KitANNEX100F100 TestsF
          ANNEX100F
          Annexin V:FITC Assay KitANNEX20F20 TestsF
          ANNEX20F
          Annexin V:FITC Assay KitANNEX300F300 TestsF
          ANNEX300F
          Annexin V:Pe Assay KitANNEX200PE200 TestsF
          ANNEX200PE
          Annexin V:Pe Assay KitANNEX50PE50 TestsF
          ANNEX50PE

          Recommended Positive Controls

            Histology Controls

              References

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