Kinetic Apoptosis Kit (Microscopy) (ab129817)

Reviews (1)Q&A (1)References (3)

Overview

  • Product name

    Kinetic Apoptosis Kit (Microscopy)

  • Detection method

    Fluorescent

  • Sample type

    Adherent cells, Suspension cells

  • Assay type

    Quantitative

  • Product overview

    Abcam's Kinetic Apoptosis Kit (Microscopy) is based on pSIVA TM technology. pSIVATM (Polarity Sensitive Indicator of Viability & Apoptosis) is an Annexin XII-based polarity sensitive probe for the spatiotemporal analysis of apoptosis and other forms of cell death. pSIVATM binding is reversible which enables researchers to detect irreversible as well as transient phosphatidylserine (PS) exposure.


    PS exposure is a hallmark phenomenon occurring early during apoptosis and persisting throughout the cell death process and it often considered to be irreversible. However, transient PS exposure is increasingly being recognized as a phenomenon in its own right and described to occur during both normal physiological processes and reversible or rescuable apoptotic/cell death events.


    The pSIVATM assays are very straightforward: add pSIVA-IANBD or pSIVA-IANBD + Propidium Iodide (PI) directly to cells or tissues, incubate and analyze.

  • Notes

    pSIVA TM is conjugated to IANBD, a polarity sensitive dye that fluoresces only when pSIVA is bound to the cell membrane. pSIVA-IANBD fluorescence is measured using conventional FITC filter sets. pSIVA-IANBD applications include flow cytometry (ab129816) and live cell fluorescence microscopy imaging.

    pSIVA™-IANBD is polar sensitive. pSIVA™-IANBD fluoresces in non-polar but not polar environments. When pSIVA™-IANBD is bound to PS it is in the non-polar environment of the membrane lipid bilayer and fluoresces. However, when pSIVA™-IANBD is not bound it is in the polar environment of the media or buffer and does not fluoresce.

    The manual contains detailed information about the pSIVA technology and protocols; please read the manual prior to beginning your experiments.

  • Platform

    Fluorescence microscope

Properties

Images

  • Kinetic Apoptosis and Necrosis Analysis of HT1080 cells
    Kinetic Apoptosis and Necrosis Analysis of HT1080 cellsThis image is courtesy of an AbReview submitted by Sarah Beckman.

    HT1080 cells were plated at a density of 2000 cells/well in 96 well plates and allowed to adhere overnight. The next day, cells were treated with dilutions of camptothecin, starting at 2500 nM, in order to determine the effect of camptothecin on the apoptotic and necrotic response of HT1080 cells. Our results show an increase over time in the percentage of GFP positive apoptotic cells (A) and PI positive necrotic cells (B). Apoptotic and necrotic cells are normalized to high contrast bright field cell counts. This increase is dependent upon concentrations of camptothecin. The accompanying figure also shows a dose response curve for apoptosis (C) and necrosis (D), demonstrating that both the apoptotic and necrotic response show a dose dependent increase following treatment with camptothecin. The dose response curve is based on 48 hours of camptothecin treatment.

  • Functional Studies - Kinetic Apoptosis Kit (Microscopy) (ab129817)
    Functional Studies - Kinetic Apoptosis Kit (Microscopy) (ab129817)

    Time-lapse images showing the progressive movement of PS exposure along axons to the cell body as detected by Kinetic Apoptosis Kit (pSIVA-IANBD). Images were taken 10-14 h (3 frames/h) after NGF removal. *Indicates where PI staining was first seen in the cell body. Black and white: pSIVA-IANBD fluorescence. Bottom panel: Merged images of phase contrast, green (pSIVA-IANBD) and red (PI) fluorescence. 100 μm scale bar. Cells were imaged with pSIVA-IANBD and PI present in the culture medium for the duration of the experiment. Figure from Kim et al 2010a.

Protocols

References

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Kinetic Apoptosis and Necrosis Analysis of HT1080 cells

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
HT1080 cells were plated at a density of 2000 cells/well in 96 well plates and allowed to adhere overnight. The next day, cells were treated with dilutions of camptothecin, starting at 2500 nM, in order to determine the effect of camptothecin on the apoptotic and necrotic response of HT1080 cells. Our results show an increase over time in the percentage of GFP positive apoptotic cells (A) and PI positive necrotic cells (B). Apoptotic and necrotic cells are normalized to high contrast bright field cell counts. This increase is dependent upon concentrations of camptothecin. The accompanying figure also shows a dose response curve for apoptosis (C) and necrosis (D), demonstrating that both the apoptotic and necrotic response show a dose dependent increase following treatment with camptothecin. The dose response curve is based on 48 hours of camptothecin treatment.
This kit was straightforward and easy to use. We performed the assay according to the manufacturer’s instructions using the lowest recommended doses of PI and pSIVA (5 and 10 ul/ml respectively). This assay was performed on a Cytation 5 Cell Imaging Multi-Mode Reader in conjunction with a BioSpa 8 Automated Incubator (BioTek Instruments, INC.).

Dr. Sarah Beckman

Verified customer

Submitted Sep 18 2017

Question

I am a PhD student having trouble finding something to measure apoptosi/necrosis in real-time.
I'd like to look at two cell types in direct co-culture and detect early and late apoptosis and necrosis in one of the cell types using real-time fluorescence microscopy.
I will probably end up doing flow cytometry, as it looks to be the most accurate way of differentiating a mixture of cells, but it would be nice to do some live-cell fluorescence imaging over several days to watch how the process unfolds.
Most apoptosis/necrosis detection assays are end-point assays and require you to stain the target cells after the induction of apoptosis. Is there anything that you can pre-stain the cells with so that you can put them in direct co-culture with another cell type that is killing them and monitor over time (say at least 3 days) for apoptosis and necrosis in the stained population alone?

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Answer

The product that is best suited to what you want to do is ab129817 for microscopy. We also have ab129816, the same reagents adapted for flow cytometry. These detect exposed phosphatidylserine. However, this occurs as an intermediate step during apoptosis, not early or late. Detection of early and late events by flow cytometry are discussed in the following two references but I am not sure if these approaches can be adapted to real-time detection via microscopy over several days.

Exp Cell Res. 2002 Jul 1;277(1):1-14. http://www.ncbi.nlm.nih.gov/pubmed/12061813

Cytometry. 1996 Jun 1;24(2):106-15. http://www.ncbi.nlm.nih.gov/pubmed/8725659

I am also not sure how easily late-stage apoptosis can be distinguished from necrosis.

For more information about the principle of the ab129816 and ab128817 assays, we recommend the following reference.

Kim YE et al. Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis. Nat Protoc 5:1396-405 (2010).

http://www.ncbi.nlm.nih.gov/pubmed/20671723?dopt=Abstract

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