[Cytometry] multiple negatives for comp in Diva?

Gert Van Isterdael gert.vanisterdael at irc.vib-ugent.be
Fri Feb 7 03:33:09 EST 2014


Hello all,

Just a small extra comment on the P3 gate that you have to draw: make 
sure to include all negative events! So put your X-axis biexponential or 
draw your gate past the left axis when the histogram is in LOG scale. 
Otherwise the calculation of the median of your neg. population will be 
incorrect and this will lead to an incorrect compensation matrix. Check 
this BD link: 
http://www.bdbiosciences.com/documents/BD_FACSDiva_setup_system.pdf Here 
they explain very good what you have to do in DIVA. (I have no 
affiliation with BD and there are lots of compensation beads on the market).

And now my question for the Flow Gurus: what if you leave a parameter 
open to have a look at the autofluorescence of your cells? Do you have 
to activate the parameter in the cytometer settings, create comp 
controls and delete the parameter out of your comp controls in the pop 
up window? Or is there another trick to do this?

Kind regards,

Gert Van Isterdael


On 06/02/2014 18:40, Cris Bare wrote:
> 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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-- 
_______________________________________________________
Gert Van Isterdael

IRC Flow Core
Unit of Immunoregulation and Mucosal Immunology
VIB Inflammation Research Center, UGent
'Fiers-Schell-Van Montagu' building
Technologiepark 927
B-9052 Ghent, Belgium

Tel : +32-(0)93313754 or +32-(0)93313578
Fax : +32-(0)93313609
E-mail : gert.vanisterdael at irc.vib-ugent.be
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