Fixed cell viability and EMA

Hello fellow Flow'ers.

I have a colleague who would like to use EMA as a viability stain on patient
blood samples, however he is unable to find a protocol on how to
reconstitute the EMA powder.

I have googled and found that people reconstitute it in either ethanol or
dimethylformamide and was wondering if anyone has a preference for either of
these?	I have read that DMF can activate PBMCs and ethanol
fixes/permeabilises the cell membrane, so how do you reconstitute EMA and at
what concentration?

I have asked everyone at our Institute to fix their human samples before
analysing them and this is causing a few headaches.

We currently have a standard 4-colour BD FACSCalibur or FACSort for analysis
(488nm and 635nm lasers with 530/30, 585/42, 670 LP and 661/16 filters).
This colleague has three necessary stains, and would like to use his "free"
channel as both a dump channel and to assess viability with EMA.  However he
colleague has read that EMA is not bright enough to use with other
antibodies in a dump channel, can anyone shed some light as to whether this
is true?  Or does anyone know of a better viability stain that works on
fixed cells?

Someone suggested you can still use PI for as a viability stain on fixed
cells, but which fixative could you use?

Cheers
Kylie


Kylie Price

Flow Cytometry Manager
The Malaghan Institute of Medical Research
The Central Services Building
Victoria University Campus
Entrance 7 
Kelburn Parade
Wellington 6242
New Zealand

Ph:	       64-4-4996914, ext 850
Fax:	       64-4-4996915
E-mail:       kprice@malaghan.org.nz
Web:	      www.malaghan.org.nz