[Cytometry] multiple negatives for comp in Diva?

Kai Witte kai.witte at med.uni-tuebingen.de
Fri Feb 7 06:19:01 EST 2014


It's even easier:
You can utilize the "universal" negative tube for most of your 
fluorescence tubes and only when you add a P3 gate will DIVA apply the 
"local" negative population included with these special tubes, e. g.:
- compensation plus beads for very highly expressed antigens,
- cells for fluorescent protein expression (GFP, etc.) or DNA/RNA dyes 
(can't any vendor come up with beads for the latter, please?),
- beads with -NH2 groups stained with amine-reactive dyes (live/dead 
discrimination dyes as well as CFSE et al.!).

For amine-reactive dyes you may need to do a titration to come up with 
suitable beads stained brighter than your samples. If stored with care 
(or as frozen aliquots), these can be utilized for extended periods like 
months (years).

I also prefer to append unstained beads (rather than "matched" control 
beads) to compensation files where needed (easier to acquire similar 
numbers of pos/neg beads), especially since I've seen supposedly matched 
pos/neg beads with quite different properties.

Best wishes,
Kai
---
On Feb 6, 2014, at 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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>
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