RE: BD fastimmune protocol

Calman Prussin (CPRUSSIN@atlas.niaid.nih.gov)
Wed, 26 Feb 1997 19:41:13 -0500

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@med.unc.edu
Phone: 919-966-4058 FAX: 919-966-0486
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