Apoptosis detection by sub-diploid population

Hello Flowers:

 

I'd like to assess apoptosis in a mouse pre-B cell line by detecting a
sub-diploid DNA population by propidium iodide on our FACSCalibur, using
the protocol below, from 
http://www.molbio.princeton.edu/facility/flowcyt/Apoptosis.html, which
seems to match the method used in a number of papers I've read.  

 

I'm first interested in detecting a normal DNA distribution in untreated
cells.	I've attached scatter, area vs. width, and count vs DNA content
plots (the parameters were: FL2 PMT 315, FL2-A AmpGain 3.57, FL2-W
AmpGain 7.21).	As shown in the histogram, I've found that the my
diploid peak is short and broad compared to those I've seen in the
literature, and I don't see much of a tetraploid peak at all.  Does this
indicate to anyone that something particular in my sample treatment or
analysis going wrong?

 

Thanks for any help!

Nancy Ogden

Research Associate

Globe Laboratories

Vancouver BC

 

PROTOCOL

1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS
+ 2% FBS; PBS + 0.1% BSA)
2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml.
3. Aliquot 1 ml cells in a 15 ml polypropylene, V-bottomed tube and add
3 ml cold absolute ethanol with vortexing. 

4. Fix cells for at least 1 hour at 4oC. 
5. Wash cells X2 in PBS. 
6. Resuspend cells in 0.5 ml PBS and add 1.0 ml DNA extraction buffer.
7. Incubate at room temperature for 5 min and centrifuge.
8. Add 1 ml of propidium iodide staining solution to cell pellet and mix
well. Add 50 ul of RNase A stock solution and incubate for 30 min at
room temperature
9. Store samples at 4oC until analysed by flow cytometry (collecting
10000 counts).

 REAGENTS:

DNA Extraction Buffer:
192 ml 0.2M Na2HPO4, 8 ml 0.1M citric acid; pH 7.8

Propidium Iodide Staining Solution: 
3.8 mM sodium citrate, 50 ug/ml PI [Sigma, P 4170]
<http://www.sigald.sial.com/safc/about_us/about.htm>  in PBS.

RNase A stock solution:
10 mg/ml RNase A [Worthington Biochemicals, RASE LS005649, LS005650]
<http://www.worthington-biochem.com/priceList/R/Ribonuclease.html>
(boiled for 5 min, aliquoted and stored frozen at -20oC).

 



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This attachment - 'count vs fl2a.JPG' -  4.50 KBytes - can be viewed at
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This attachment - 'scatter.JPG' -  7.41 KBytes - can be viewed at
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