[Cytometry] Population seen in the APC channel

Dravid, Gautam GDravid at mednet.ucla.edu
Fri Jul 10 12:52:48 EDT 2009


Hi,

Our lab works with cord blood CD34+ cells and drive lymphoid differentiation to CD19+ B cells on mouse stroma (OP-9 or MS-5). We culture the CD34+ cells for 3 weeks on stroma and look for CD19 expression.

When we used CD19-APC we saw a "Distinct" population of CD19+ cells when using a FACSAria, with a band pass filter of 660/20. for the APC detector.

The problem is when we run JUST UNSTAINED cells (WITHOUT ISOTYPE ANTIBODY) we see the exact same "distinct" what appears like a positive population in the APC detector!! and it LOOKS VERY REAL.

We tried the following

1) If we run the stroma alone the population disappears

2) If we run the CB alone (grown under stroma free conditions) the population disappears.

So the population is seen only when CB is present on stroma. Also the further the time you culture the CB more is the population seen, e.g. for  2 weeks < 3 weeks over <5 weeks culture.

Has anyone encountered similar problem, and what's the solution besides avoiding APC?

Thanks

Gautam Dravid
Assistant Researcher
UCLA
615 Charles Young Drive South
OHRC bldg, 547
Los angeles, CA -90095.
Tel: 310-825-8466

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