I am using the SGBS preadipocyte cells that are differentiated with a mix of factors to mature adipocytes (up to 14 days). I am trying to check if my cells turn apoptotic when adding one or more substances to the media.
Untill now, we have been using the Nicoletti essay as described in the FIRST Nicoletti publication (1997 was it?). Problem is, I get a high amount of false positive (up to 30%) apoptosis in my media control which leads me to think that this method might not be the ideal one.
Do you have any alternatives that I could try? If so, which protocol do you suggest?
Edit: I want to try and avoid flow cytometry. I grow my cells in 12-well plates (they differentiate best) but they are also fine in Chamber Slides. So flourescent microscopy is the best....
I want to be able to determine if the death is apoptosis or necroptosis.
Do you have flow cytometry or fluorescent microscopy for the detection of apoptosis?
1-I suggests that you can to use colorimetric assay for measurement of Caspae-3 activity in your extracted suspension.
2- DNA Laddering test is the another suggested method that perform by Gel electrophoresis
3-If you want to evaluate the expression markers in mRNA level such as Bax, cox, I suggests that used from RT-PCR or Real Time PCR strategies.
Best
Annexin V kits are good indicators of apoptosis and/or late apoptosis. They work great on Flow Citometry but can be used under fluorescence microscopy aswell.
Two approaches: TUNNEL staining or activated caspase 3 immumnofluorescence staining. Activatecd caspase 3 expression can also be analyzed using Western Blot.
I would suggest an annexin -v assay or activated caspase 3 levels using western blot.
Analyse gene expressions of Bcl-2, BAX, Caspase 3.
Fluorescent microscopy also useful.
i think FLICA assay is a useful tool for such cases, AO/ EtBr staining is informative as well.
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Jessica, you can acquire the cells by contacting our boss Prof. Dr. Martin Wabitsch and ask him to send you the cells
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