about ICS

Milton Maciel Junior mmaciel at usp.br
Thu Jun 28 16:25:24 EDT 2007


Dear all,
a couple of technical questions, specially for people running  
intracellular staining. I just read a manuscript ahead printing that  
brought me some questions about the methodology for intracellular  
staining.
First: is it possible, or what is the result, if the monensin (or  
brefeldin) is added at the same time of the stimulation (protein,  
peptides etc) and left ON, instead of  5 hours "after" de ON  
incubation with the stimuli? (the cytokines in question are IFN, TNF  
and IL-10, in human samples of frozen PBMCs)

Second: what if, instead of labeling the cells with all surface  
markers (CD3, -4, -8, -27, -62 etc) first, and then begin the  
permeabilization step for the intracellular staining, to label	
everything at the same time, i.e. begin with the permeabilization and  
then add all markers (surface and intracellular cytokines) at the same	
time in one single step?

I am putting together a 10- color panel and I am trying to optimize  
the labeling procedure, making it shorter...

Thanks a lot for all comments and answers...
MM

Milton Maciel Junior, PharmD, Ph.D
University of Maryland
School of Medicine
Center for Vaccine Development
Baltimore-MD-USA-21201
Phone: (410) 706-7376





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