[Cytometry] multiple negatives for comp in Diva?

David McDonald David.McDonald at newcastle.ac.uk
Fri Feb 7 06:51:01 EST 2014

Hi Carol,

Just to add to Cris's message. If you have a very high transfection efficiency so that you don't have a negative histogram for your transfectants then either mix unstained cells into your transfectants or acquire and append  some unstained cells into the "GFP" compensation control tube (you need a minimum of 5000 events for the autocomp to work). 

We take the same approach to for compensating for viability dyes such as DAPI.

Best wishes,

Dr D McDonald

Flow cytometry core facility
Newcastle University, UK

>-----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?
>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.
>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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