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Strange signal shift

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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Jul 17, 2018 6:23 pm

Re: Strange signal shift

Hi Sheila,

I agree, I think fixation is a strong possibility.

Have you tried doing a PFA fix after surface stain (ie, before eBio perm/fix)?

Have you tried 2% rather than 1.6%? Just in case you're getting some variation in your residual volume after flicking/aspirating, and therefore your *true* final PFA concentration is lower than you thought?


Mike
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mccars

Participant

Posts: 4

Joined: Wed Jun 15, 2016 10:56 am

Post Thu Jul 26, 2018 4:16 pm

Re: Strange signal shift

Hello,
I thought I’d post an update on this problem.

Since last posting I’ve included an additional fixation steps and run an experiment in parallel with colleagues using the same technical healthy control.

They used their reagents, and I opened fresh standard reagents where possible (including RPMI media; MaxPar cell staining buffer; MaxPar Water; FOXP3 staining buffer kit; 16% PFA and MaxPar FixPerm borrowed from colleagues).

I didn’t carry out any stimulation or barcoding steps.

I stripped the panel down to a small selection of the troublesome ab only:
142Nd IL-4 145Nd CD4 146Nd CD8
150Nd IL-22 154Sm CD3 173Yb HLADR
The samples were also allowed to sit in water for more than an hour and then run again.
The samples were run for more than 1 hr.

The strange signal was not apparent in either experiment.
This indicates these particular antibodies are ok.

So, I ran my samples again, this time using the full panel and including the barcoding and stimulation steps. I also had an unbarcoded and unstimulated control, that was stained immediately.
I used the same PFA fixation steps for both samples i.e. a 10min fixation in 2% PFA (as recommended by Mike) followed by resuspension and incubation overnight in MaxPar fix/per with 1.6% PFA added. The PFA vial was cracked open immediately prior to fixation.

The strange signal showed up again, but really only within the 150Nd IL22 channel within in the sample incubated for 4hrs (both at rest and stimulated) plus barcoded, but not in the control; however the barcoded sample was run for 1 hr 45 mins, and the control only for 15 mins due to cell number.

My next steps will be to investigate if this is due to: a full panel of ab; barcoding; or stimulation.
I’m also going to try the pre-foxp3 fix/perm fixation step recommended by Mike.
I have also sent Zach Bjornson one of the IMD files.


Any thoughts?

And thanks for your help so far!
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