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Overnight Background Increase

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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Tue Dec 24, 2013 12:17 am

Overnight Background Increase

Dear All,

I've stained PBMC with surface markers. Then I washed and fixed with 1.6% paraformaldehyde. The 16% PFA was fresh from a break open vial, and diluted with Ca++/Mg++ free PBS. Half of the sample was collected on the Cytof, and the other half was placed at 4C for collection the next day. In comparing the data, the only differences I saw were in the background. The overnight sample had increased nonsense populations like CD3+ and CD20+. Is this to be expected? Is there a way to avoid it?
I've attempted to attach a pdf.

Ian

overnight compare.pdf
(113.78 KiB) Downloaded 396 times
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Tue Dec 24, 2013 5:27 am

Re: Overnight Background Increase

Which channels did you use for these markers?
Could it be environmental contaminants? (Pb, Ba)
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antonio

Contributor

Posts: 20

Joined: Wed Dec 04, 2013 3:05 pm

Post Thu Dec 26, 2013 9:06 pm

Re: Overnight Background Increase

How cell were stored during the night? In which buffer?
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Mon Jan 06, 2014 10:22 pm

Re: Overnight Background Increase

The cells were stored at 4C overnight in 1.6% PFA in PBS. I don't think it could be environmental contaminants as those would show up in both samples.
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antonio

Contributor

Posts: 20

Joined: Wed Dec 04, 2013 3:05 pm

Post Mon Jan 06, 2014 10:31 pm

Re: Overnight Background Increase

Are your cells simultaneously positive for CD20, CD3, CD16, HLA-DR? If this is the case we observed such a population positive for everything sometime. I do not know whether it increase during the ON storage.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Jan 06, 2014 11:25 pm

Re: Overnight Background Increase

Have you tried just leaving the cells in PBS overnight, rather than PFA+PBS?

Basically, I wonder if you're just starting to crosslink cells to each other.


Just to make sure I'm understanding your workflow:

1. Thaw cells and wash.
2. Stain with surface markers. wash.
3. Fix with PFA/PBS.
4. Split in two: 4a and 4b.
5. Wash 4a with PBS, then MilliQ water and run on CyTOF. Leave 4b in the same PFA from Step 3/4, overnight, 4C.
6. Wash 4b with PBS, then MilliQ water and run on CyTOF.

Is this correct? Or did you wash all the cells with PBS, then split, and put 4b *back into* PFA/PBS?


Mike
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ianfrank

Contributor

Posts: 29

Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Tue Jan 07, 2014 5:10 pm

Re: Overnight Background Increase

Your steps 1-6 are correct. I did not wash and put back into PFA.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Jan 07, 2014 6:54 pm

Re: Overnight Background Increase

Then I would be curious what your results would be if you *did* wash away the PFA and then leave in PBS overnight to run Day 2.

If it's a cell crosslinking issue due to PFA overnight, then doing that should make your "weird cell"/background number remain the same as Day 1's short-fix sample.

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