[Cytometry] multiple negatives for comp in Diva?

Cris Bare flowmail at verizon.net
Fri Feb 7 08:52:00 EST 2014


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
>> 91 N. Eagleville Rd
>> Storrs, CT 06269-3149
>> 
>> Phone (860) 486-3080
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