Overview

  • Product name
    Autophagy/Cytotoxicity Dual Staining Kit
    See all autophagy kits
  • Sample type
    Adherent cells
  • Assay type
    Quantitative
  • Species reactivity
    Reacts with: Mammals, Other species
  • Product overview

    Autophagy/Cytotoxicity Dual Staining Kit (ab133075) provides a convenient tool for studying the regulation of autophagy and cytotoxicity at the cellular level. The kit employs monodansylcadaverine (MDC), a fluorescent compound that is incorporated into multilamellar bodies by both an ion trapping mechanism and the interaction with membrane lipids, as a probe for detection of autophagic vacuoles in cultured cells. Propidium iodide (PI) is used as a marker of cell death. Tamoxifen, a known inducer of autophagy, is included as a positive control. This kit provides sufficient reagent to effectively treat/stain 960 individual wells of cells when utilized in a 96-well plate format. Lower density plates will still require approximately the same amount of reagent on a per plate basis. Therefore, up to 10 plates worth of cells can be examined irrespective of the number of wells/plate (this is not the case for protocols that use non-adherent cells).

  • Notes

    Autophagy is a critical cellular process that involves the degradation and digestion of intracellular components by the lysosome. This process not only enables cells to efficiently mobilize and recycle cellular constituents, but also prevents the accumulation of damaged organelles, misfolded proteins, and invading microorganisms.

    Autophagy is a multi-step process that begins with the sequestration of cytoplasmic organelles and proteins. These cellular components are sequestered by a double membrane, forming an autophagosome. The autophagosome then fuses with a lysosome to form an autolysosome, where the cellular material is then degraded. Normal autophagy is essential for survival, differentiation, development, and homeostasis. Dysregulation of autophagy has been implicated in cancer, infection, aging, and degenerative diseases.

    While autophagy most often acts to promote cell survival in response to stress, it can also promote cell death. The relationship between autophagy and apoptosis is complex. The two pathways share common stimuli and components, and can regulate the activity of each other. However, the specific factors and mechanisms that dictate the choice between autophagy and apoptosis remain unclear.

  • Platform
    Fluorescence microscope

Properties

Images

  • HepG2 cells were seeded in a 96-well plate at a density of 5 x 104 cells/well in EMEM culture medium and incubated O/N at 37°C. The next day, cells were treated with increasing concentrations of tamoxifen and incubated O/N. On the third day, cells were stained with PI and MDC (as described in the assay protocol) and fluorescence was quantified using a plate reader.
    Top panel: Tamoxifen treatment increases MDC fluorescence intensity, indicating that Tamoxifen treatment leads to an increase in autophagy in HepG2 cells.
    Bottom panel: Tamoxifen treatment does not cause an increase in PI staining, indicating that at the concentrations used in this experiment, tamoxifen does cause cytotoxicity in HepG2 cells.
  • HepG2 cells were seeded at a density of 5 x 104 cells/well and incubated O/N at 37°C. The next day, cells were treated with either vehicle (panel A & B) or 10 µM of tamoxifen for 24 hours. On the third day, cells were stained with PI and MDC as described in the assay protocol.
    Panel A: MDC staining of HepG2 cells treated with vehicle. There is a basal level of autophagy, indicated by faint silver dot staining of autophagic vacuoles. Panel B: PI staining of HepG2 cells treated with vehicle. There are few dead cells with only background staining of propidium iodide.
    Panel C: MDC staining of HepG2 cells treated with 10 μM Tamoxifen. There is a clear increase in fluorescence intensity and number of autophagic vacuoles compared to the control cells treated with vehicle. Panel D: PI staining of HepG2 cells treated with 10 μM Tamoxifen, shows similar staining pattern to that of cells treated with vehicle.

Protocols

References

This product has been referenced in:
  • Wu C  et al. Defective autophagy in chondrocytes with Kashin-Beck disease but higher than osteoarthritis. Osteoarthritis Cartilage N/A:N/A (2014). Read more (PubMed: 25168363) »
See 1 Publication for this product

Customer reviews and Q&As

Answer

This kit is designed for suspension cells. The point of the centrifugation is to pellet the cells to aid in transfer of staining and wash solutions without losing cells. If you do not have a plated centrifuge, then staining can be performed in microcentrifuge tubes. Use care to avoid aspirating cells during the staining and washing.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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