[Cytometry] multiple negatives for comp in Diva?

Ian Titley Ian.Titley at icr.ac.uk
Fri Feb 7 04:29:18 EST 2014


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
> Fax (860) 486-5005
> 
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