[Cytometry] multiple negatives for comp in Diva?

Smith, Robert Robert.Smith3 at pfizer.com
Thu Feb 6 12:11:38 EST 2014


Hi Carol,
You don't want to use the cells as the universal negative. The different autofluorescence between the beads and cells will mess up the comp calculation.
You can, however, run the negative and positive populations of cells or beads in the same tube and Diva will then reference the correct negative when compensating.
In the Create Compensation Control window uncheck the box that says "include separate unstained control." Diva will then look for a negative and positive population in each tube and calculate compensation appropriately. Just check that the auto gates correctly identify the populations.
You will use up more negative beads this way and have to add them to all your bead controls but it makes things easier down the road.
Good luck,
Rob Smith

________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] on behalf of Carol Norris [carol.norris at uconn.edu]
Sent: Thursday, February 06, 2014 11:40 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] multiple negatives for comp in Diva?

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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