[Cytometry] multiple negatives for comp in Diva?

Ian Titley Ian.Titley at icr.ac.uk
Fri Feb 7 10:02:34 EST 2014


If Carol is using PE and GFP excited from say a blue (488nm) laser only whereby she expects a great deal of "spill-over" from GFP into PE the aim of her compensation would be to adjust the GFP positive population to show the same fluorescence value as the negative (autofluorescence) in the PE channel by whichever statistical measure of central tendancy. Similarly she would want the same calculation for the PE to GFP spill-over (the correction would probably be of a lower value). If two types of particle are used, say beads and cells, (one for each channel) and their autofluorecence differs I cannot see how this can be done accurately?

Ian

-----Original Message-----
From: Cris Bare [mailto:flowmail at verizon.net] 
Sent: 07 February 2014 14:45
To: Cris Bare
Cc: Ian Titley; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] multiple negatives for comp in Diva?

Oh, I see where I'm not being clear. 

The positive and negative cell/bead should match within the same tube. I was saying channel and that can be misconstrued. 

Since comp controls should be one color only, that tube (think of it as one data file) will have both the positive stained events and the denominator/baseline/control/negative events. 

Having both positives and negatives in the same tube/datafile works in all software package autocompensation wizards (Flowjo, FCSExpress, DiVa, etc).

-cbb

On Feb 7, 2014, at 6:18 AM, Cris Bare <flowmail at verizon.net> wrote:

> Nope. 
> 
> The only signal that matters is the fluorochrome. By matching the intrinsic signal between the positive and negative in the same channel you essentially set that intrinsic as baseline and any signal above that is from the fluor. That fluor signal is then used to calculate the slope of the spillover regression. 
> 
> On Feb 7, 2014, at 6:03 AM, Ian Titley <Ian.Titley at icr.ac.uk> wrote:
> 
>> Yep. However if Carol is using cells for the GFP channel and beads for say the PE channel will the fact that the autoflourescence of the two types of particle does not match in both channels impact the compensation between those channels?
>> 
>> Ian
>> 
>> -----Original Message-----
>> From: Cris Bare [mailto:flowmail at verizon.net]
>> Sent: 07 February 2014 13:52
>> To: Ian Titley
>> Cc: Carol Norris; cytometry at lists.purdue.edu
>> Subject: Re: [Cytometry] multiple negatives for comp in Diva?
>> 
>> Let me see of I can beat Mario this time. 
>> 
>> The autofluorescence ( AKA intrinsic fluorescence) should match between the positive population and the negative population within the same detector channel. 
>> 
>> For example, if you capture PECy7 on a bead, use the corresponding bead negative, but if you use GFP the negative should be the same cell type non-expressor. 
>> 
>> That's why I suggest manually drawing the "P3" negatives gate in the histogram instead of using the universal unstained negative. 
>> 
>> -cbb
>> 
>> On Feb 7, 2014, at 1:29 AM, Ian Titley <Ian.Titley at icr.ac.uk> wrote:
>> 
>>> Will this work if the "autofluoresence" intensity of the "negative" beads and "negative" cells are significantly different within the same compensation experiment? I think this might be what Carol is concerned about? Can Carol use antibody labelled beads as "positives" and unlabelled cells as the universal negative control within the same compensation run?
>>> 
>>> Best wishes
>>> 
>>> Ian
>>> 
>>> 
>>> Ian Titley PhD
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