[Cytometry] compensation

Nicole Rund nicole.rund at biotec.tu-dresden.de
Tue Mar 15 05:33:49 EDT 2011


Dear all,

I had the same problen (mix of bead-based and cell-based samples in one 
compensationsmatrix).
The BD-applicationsspecialist told me, that I can put the P2-gate on the 
negativ and create a P3-gate on the positiv ones. Then the Diva software 
would use the P2gate (and not the universal unstained) to calculate the 
compensation.
So now I use the beads as universal unstained and use P2 as positiv-gate 
for my bead-based samples. For the CFSE sample I mix stained and 
unstained cells and gate negativ cells with P2 and positiv with P3. For 
me it look good in this way, but I`m not an expert.

Any annotation? Is there anyone with expertise in this method and 
noticed "pitfalls"?
Thanks.

Best,
Nicole

Am 3/15/2011 12:52 AM, schrieb Mario Roederer:
> Again, I'd like to stress that there are only 3 rules to achieve proper compensation:
>
> For each color in any given panel:
>
> (1) Your compensation control color must be matched to your experimental color
> (2) The positive control must be at least as bright as your experimental sample
> (3) The positive control for your compensation sample, when unstained, must have the same background fluorescence as the negative control for that color.
>
> That's it.
>
> You'll note that there is nothing about PMT voltages, filters, autofluorescence, cell type, beads, day of the week, phase of the moon, or mood of the operator involved.
>
> All reasonable problems with compensation can be traced to a violation of one of these 3 rules.
>
> I will note that (current) DiVa software enables a common violation of rule #3, by only allowing only one universal negative -- thus preventing proper compensation when a mixture of bead-based and cell-based compensation controls are used.  Better-designed software does not make this fundamental mistake.
>
> mr
>
> (PS, I left out one rule: that you use the same instrument fluorescence settings for all samples and compensation controls.  Violating this rule is more fundamental than anything compensation-related; including it as a rule would be akin to specifying that you must turn on the instrument before you run any controls.  This would be what I would refer to as an "unreasonable problem" with compensation.)
>
>
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