[Immunology] Adding apoptosis marker to cell cycle analysis

Hanlon, Paul via immuno%40net.bio.net (by phanlon from Standardprocess.com)
Wed Aug 20 10:57:22 EST 2008


I have been conducting research on the effects of phytochemicals and
cell cycle and apoptosis and I would like to find a way to run both
assays on the same cells. 

 

I am currently using the NIEHS protocol for measuring cell cycle with PI
and an anti-BrdU FITC antibody. I would like to keep this method since I
really like the added confidence of S phase determination that the BrdU
tagging gives me. I am looking for an apoptosis marker to add to this,
does anyone have anything they know or would hypothesize would work? I'm
thinking either a caspase substrate or some sort of antibody to an
apoptosis-related protein. 

 

My current protocol gives me excellent cell cycle staining and it
involves:

1.	Adding BrdU to the cells for 40 minutes
2.	Fixing the cells with ethanol
3.	Solubilizing the cells with 2N HCl + 0.5% Triton-X 100
4.	Overnight staining with the anti-BrdU antibody (BD)
5.	Overnight staining with PI

 

I then read on the flow with a FACSCalibur (FL1 = FITC, FL3 = PI).

 

I'm willing to try another DNA stain if it helps to free up a detector,
but I'd really like to leave the rest of the method the same because it
is working so well. I'd like to add something like a caspase substrate
which I could incubate at the same time as the BrdU, but I'm afraid of
the substrate disappearing when the cells are solublized. 

 

I'm willing to try anything, so educated guesses would be appreciated.
Thanks

 

Paul Hanlon

 

Manager of Discovery Science

Standard Process, Inc.

1200 West Royal Lee Dr.

Palmyra, WI 53156

Phone: 262-495-6493

Fax: 262-495-6399

phanlon from standardprocess.com

 



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