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 ---------- 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 ======================================================================= ==== 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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