about ICS

Hollier, Mark J. (CDC/CCID/NCZVED) (CTR) etu4 at CDC.GOV
Fri Jun 29 15:04:06 EDT 2007


HI Milton,

I am not able to help you much with question 1, but my thoughts (and
these are just theoretical thoughts) is that if you add Monensin at the
same time as the stimulant that you will see no increase in protein
production. This would theoretically be due to the protein
synthesis/transport being shut off before any translation of transcripts
could occur. As I said, I have no experience with this, just my
thoughts.

For the second question, I can provide some help. I have tried to
perform surface staining of some molecules (CD3, CD56, CD16, CD19) after
fixation and permeabilization. There are 2 problems with this:

1. If you permeabilize the cells and then try surface staining, you will
also be staining the intracellular (recycled and newly made proteins)
proteins that are not on the surface. Hence it will not be surface
staining, it will be total staining.

2. Cross-linking surface epitopes before staining can result in the
antibody no longer binding to the epitope. I found this to be true for
CD19, CD56, CD3, and CD16.

Hope this is of help,

Mark.

Dr Mark Hollier
Centers for Disease Control and Prevention,
Coordinating Center For Infectious Diseases,
National Center For Zoonotic, Vector-Borne, & Enteric Diseases,
Division Of Viral & Rickettsial Diseases,
Chronic Viral Diseases Branch,
Atlanta, GA, 30329
USA
E-mail:etu4 at cdc.gov
Tel: 404-639-2276
Fax: 404-639-3540

-----Original Message-----
From: Milton Maciel Junior [mailto:mmaciel at usp.br] 
Sent: June 28, 2007 16:25
To: cyto-inbox
Subject: about ICS

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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