[Cytometry] multiple negatives for comp in Diva?

Cris Bare flowmail at verizon.net
Fri Feb 7 09:18:50 EST 2014


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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>> 
>> -----Original Message-----
>> From: cytometry-bounces at lists.purdue.edu 
>> [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Cris Bare
>> Sent: 06 February 2014 17:41
>> To: Carol Norris
>> Cc: cytometry at lists.purdue.edu
>> Subject: Re: [Cytometry] multiple negatives for comp in Diva?
>> 
>> Carol,
>> 
>> It's easier than you think. 
>> 
>> In DiVa on the comp controls worksheet in the fluorochrome specific histogram where you adjust gate P2 to select the positive population, you can draw a P3 gate on the negative population and DiVa will use P3 preferentially for calculation. 
>> 
>> That's a word sentence. 
>> 
>> 1. Include negatives in each comp control tube. 
>> 2. Add a P3 gate on the negatives in the same histogram as P2 positives. 
>> 3. Calculate. 
>> 
>> This is why DiVa has the checkbox when you create your comp tubes to include or not include a universal negative. 
>> 
>> Cheers!
>> -cbb
>> 
>> On Feb 6, 2014, at 8:40 AM, Carol Norris <carol.norris at uconn.edu> wrote:
>> 
>>> Hi Flow Gurus,
>>> 
>>> We have lots of folks combining GFP-expressing cells with antibody labeling, and I’d like to get more of them using comp beads for the antibody controls. There doesn’t seem to be a way to use GFP-negative cells for the GFP comp control and unstained beads for the antibody comp controls in Diva.  Are unlabeled cells OK to use as a universal negative?  The other option would be to transfer the data to FlowJo to get a comp matrix, and then transfer the comp values back into Diva.
>>> 
>>> Thanks,
>>> Carol
>>> 
>>> 
>>> Carol E. Norris, Ph.D
>>> Facility Scientist
>>> Flow Cytometry/Confocal Microscopy Facility Biotechnology/Bioservices 
>>> Center University of Connecticut Unit 3149
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>>> 
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