Multi-Color Flow on Patient Samples

Calman Prussin cprussin at niaid.nih.gov
Tue Jan 23 13:28:56 EST 2007


Elizabeth, 

In regard to your question: Essentially the question is can you compare a
sample from a patient at multiple points over the course of a year and have
it actually mean anything?

What I have done in several clinical studies with serial analysis extending
over 3-6 months is at each time point to fix the (unstained) cells in 4% PFA
and freeze them at ­80. When the study is finished, all samples from a given
subject are stained and analyzed simultaneously. This requires use of mAb
clones that recognize paraformaldehyde stable epitopes, but for most Ags,
that is not a problem.

We have used this approach in both of the papers below (4 color) as well as
a current manuscript (6 color):

Foster, B., D. D. Metcalfe, and C. Prussin. 2003. Human dendritic cell 1 and
dendritic cell 2 subsets express FcepsilonRI: Correlation with serum IgE and
allergic asthma. J Allergy Clin Immunol 112:1132-1138.

Prussin, C., D. T. Griffith, K. M. Boesel, H. Lin, B. Foster, and T. B.
Casale. 2003. Omalizumab treatment downregulates dendritic cell FcepsilonRI
expression. J Allergy Clin Immunol 112:1147-1154.

We use the fixation protocol from the ICCS chapter in CPI: Foster B and
Prussin C. 2002. Unit 6.24, Detection of intracellular cytokines by flow
cytometry. In: Current Protocols in Immunology, J.E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober and R. Coico, eds. John
Wiley & Sons, New York, NY.
 
Calman


From: "Kraus, Elizabeth" <elizabkr at BaylorHealth.edu>
Date: Fri, 19 Jan 2007 16:47:19 -0600
To: cyto-inbox
Conversation: Multi-Color Flow on Patient Samples
Subject: Multi-Color Flow on Patient Samples

For those of you out there monitoring patient samples using multi-color flow
cytometry (6+ fluorophores), I would like to know of any obstacles and/or
considerations I should be aware of.  Specifically, I¹m looking for
guidelines (if there are any) on titrating multiple antibodies,
combining/pooling antibodies, standardization of reagents over multiple
sampling sessions, etcŠ Essentially the question is can you compare a sample
from a patient at multiple points over the course of a year and have it
actually mean anything?  Any thoughts would be appreciated.
 
Elizabeth T. Kraus

Manager, Flow Cytometry core Facility

Baylor Institute for Immunology Research

3434 Live Oak, Suite 200.1

Dallas,  TX.  75204

214-820-3586

elizabkr at bhcs.com

 

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