[Cytometry] Standardization of background(neg) cells

Ruud Hulspas rhulspas at cytonomest.com
Tue Feb 9 12:36:35 EST 2010


I would start with standardizing the cell labeling protocol (this is 
harder than you may think).
Second, don't 'set' the negative cell population because the 
autofluorescence may vary.
The performance of the machine is best characterized using standardized, 
non-biological particles.
If you have a machine on which the PMT voltages are not pre-set (like 
most flow cytometers), I would set them on 'Blank' particles.

Ruud


Houston, Jim wrote:
> 4 people are each asked to analyze a simple exp.  This is a one color CD3 APC Exp.  Two tubes are used. Tube #1 is Cells alone/Isotype.  Tube #2 is the actual test sample labeled with CD3 APC.  The investigator wants not only the % pos, but also the mean channel of fluorescence.
>
> Each operator then uses the same tubes (samples not split) to setup a document and produce the needed results.
>
> When comparing the results, three operators produce similar %Pos.  All produce different Mean Channel values of wide degrees.
>
> The question is.... What procedure must each use when setting up the instrument that will insure the mean values of the positive pop are within a reasonable difference?  Obviously a visual diff means dramatic problems in the way an instrument is setup.
>
> This seems like such a basic flow question, but my guess is that most people just "eyeball" where they set the negative population.
>
> This is a machine independent question.
>
>
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*Dr. Ruud Hulspas, Ph.D.*
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