RE: Programmable water baths, pH & Cytokine Flow Cytometry

We have used this method with RPMI supplemented with 5mM HEPES. We found that if you do
the incubation with Brefeldin A in a CO2 incubator with the tube tops loose, then snap
the tops tight to put them in the waterbath, the pH seems to be maintained well. It's a
great way to do a 12hr incubation

Adrian Robins

-----Original Message-----
From: Calman Prussin [mailto:cprussin@niaid.nih.gov] 
Sent: 05 June 2008 19:58
To: cyto-inbox
Subject: Programmable water baths, pH & Cytokine Flow Cytometry

A few years back Holden Maecker and the group at BD published a number of reviews and
white papers that made use of programmable water baths to culture T cells for
intracellular cytokine staining. We have an experiment in which we'd like to do a night
time 6 hour incubation in the water bath, followed by a cool cycle, after which we come
into work in the early AM, stain the cells, and then bead and flow sort them.

Question: 
In these papers they used RPMI complete medium during the water bath incubation. I would
think that at atmospheric CO2, in carbonate buffered media such as RPMI the pH would
increase and the PBMC would not be "happy".
I have heard of people using HBSS or a HEPES buffered complete medium, but cannot find
any references to such.

--> Does anyone have experience using non-CO2 buffered media for 
--> activating
T cells for intracellular cytokine staining?

Thanks,

Calman
--
_______________________
> Calman Prussin
> Laboratory of Allergic Diseases
> National Institute of Allergy and Infectious Diseases/ NIH Bethesda, 
> MD
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