BD fastimmune protocol

Calman Prussin CPRUSSIN at atlas.niaid.nih.gov
Wed Feb 26 19:41:13 EST 1997


I am concerned that by only collecting 3000 CD3+ cells you will be 
generating less than the most robust numbers for IL-4 frequencies. The 
significance of a frequency is proportional to the number of events 
you acquire. David Parks has a table in the FACS chapter he wrote for 
Paul's Fundamental Immunology, 2nd edition. By only collecting 3000 
events, a value of 5% could be anything from 4 to 6%. By increasing 
that to 50,000 events, you narrow the range to 4.7% to 5.3%.

_______________________
Calman Prussin
Laboratory of Allergic Diseases
NIAID/ National Institutes of Health

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From: 	Michelle N Fiordalisi
Sent: 	Wednesday, February 26, 1997 9:39 AM
To: cyto-inbox
Subject: 	Re: BD fastimmune protocol


Since I've had several requests for a workable protocol for 
intracellular
cytoking staining, I'd lot I'd post it for every one.

The procedure I've been using is based on the BD protocol published in 
their Application Note 1.

[text deleted]

I then acquire 3000-5000 events in a CD3+ gate and analyze by
quadrant analysis (CellQuest) of dot plots.  I calculate % positivity 
using BD's formula:  (Activ. sample - activ. isotype control) - ( 
Unstim.
sample - Unstim. isotype control).

-- Michelle

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Michelle N. Fiordalisi, Ph.D.
Clinical Immunology Fellow
University of North Carolina Hospitals
email: mnfiord at med.unc.edu
Phone: 919-966-4058  FAX: 919-966-0486
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