Intracellular % Formula

Xi-Feng Yang xyang at pharmingen.com
Fri Feb 28 14:48:07 EST 1997


RE>>Intracellular % Formula
I usually calculate % positivity using this formula:  fluorochrome conjugated
antibody blocked by un-conjugated isotype control - the staining blocked by un-conjugated
antibody.

Xifeng Yang

--------------------------------------
Date: 2/27/97 7:36 PM
To: cyto-inbox
From: DODONNEL at svherc.ucd.ie



Michelle Fiordalisi wrote

"I calculate % positivity using BD's formula:  (Activ. sample - activ. isotype
control) - ( Unstim. 
sample - Unstim. isotype control)."

I would be interested in other intracellular flow-ers opinion of this formula:
do you use it 
widely?  

 I have a problem with subtracting the unstimulated sample values.  I presume
tnat this is 
intended to allow for non-specific intracellular staining that can occur after
permeabilisation.   
However, in some ex vivo situations where effector lymphocytes are already
activated a small 
percentage of even unstimulated cells may be clearly positive relative to their
isotype control.    I 
doubt that it would occur in peripheral blood lymphocytes but I have certainly
seen it for IFN-g 
in activated BAL cells in pulmonary disease.  It occurred in situations where
the clinical disease 
was highly active.    By subtracting this percentage of cells from that in the
stimulated sample
one might be underestimating the total % of cytokine-expressing cells.

What do the erudite people think?

Dearbhaile O' Donnell,
University College Dublin


 

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Subject: Re: Intracellular % Formula
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