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Quick apoptosis detection - (Jan/04/2007 )

Hi,

I would like to do a quick analysis of apoptosis in my HEK293 cells. Can anyone advise me on a kit that is reliable and fast? I have looked though those commercially available and as there are so many I am not sure where to start!

Many thanks.

-Debz-

try a DAPI stain, u will b able to notice the apoptotic bodies if the cells underrgo apoptosis.

-scolix-

I really like the Roche Cell death assay: you lyse cells, spin, and do a sandwich elisa on supernatants. Very easy and takes 3 hours. Expensive though, 405$ for 96 assays. Good luck!

-nancyd-

QUOTE (Debz @ Jan 5 2007, 12:57 AM)
Hi,

I would like to do a quick analysis of apoptosis in my HEK293 cells. Can anyone advise me on a kit that is reliable and fast? I have looked though those commercially available and as there are so many I am not sure where to start!

Many thanks.


the fastest I know is light microscopic inspection for zeiosis (membrane blebbing);

a combined staining of annexin V-GFP and propidium iodid is for fluorescence microscopy, and discriminated between early apoptosis and late apoptosis or necrosis

-The Bearer-

hi,

a DiOC6(3) staining for 15 minutes
very cheap!
QUANTIFICATION by cytometry

Sebastien

-tryptofan-

QUOTE (tryptofan @ Jan 5 2007, 11:03 AM)
hi,

a DiOC6(3) staining for 15 minutes
very cheap!
QUANTIFICATION by cytometry

Sebastien



Is this a membrane staining method? How is that be apoptosis-specific? Can I get a protocol from you? Thanks ahead!

-genehunter-1-

hi genehunter-1,

you are right, DiOC6(3) is also a probe for the endoplasmic reticulum, but when used at a specific concentration, it serves as a sensor for mitochondrial membrane potential variations (see the paper below)

1. Ozgen, U., Savasan, S., Buck, S., and Ravindranath, Y. (2000) Comparison of DiOC(6)(3) uptake and annexin V labeling for quantification of apoptosis in leukemia cells and non-malignant T lymphocytes from children, Cytometry 42, 74-78.


this is my protocol:
1. Incubate the cells is complete medium in the presence of apoptosis inducer agents for the desired time
2. Wash the cells once with PBS (if adherent, I use citric saline, see topic http://www.protocol-online.org/forums/inde...howtopic=17456)
3. Inucate the cells in 40 nM DiOC6(3) (for c. 500,000 cells) in 300 ┬ÁL PBS, in the incubator (37C/CO2) for 15 minutes
4. Pass the cells directly, with no other washing step, to cytometer; FSC/SSC/FL1 active. Apoptotic cells are DiOC6(3) "low", and I usually place the non-apoptotic peak at 1E3.

Sebastien_

-tryptofan-

Fastest I would say is light microscopy to see apoptotic bodies/blebbing of cells. Apoptotic cells will look like popcorn. After this you can use other techniques, there are many.

-exploresci-