FAQ  •  Register  •  Login

Low resolution signal

Forum rules
Please be polite and civil. We know that troubleshooting is vexing...
<<

narulam

Participant

Posts: 9

Joined: Sun Mar 29, 2020 10:28 am

Post Thu Dec 24, 2020 4:35 am

Low resolution signal

Hello fellow user,

I recently performed a cytof run that shows poor resolution between positive and negative populations mostly due to high intensity of the negative population than usual (Run 2 attachment). I'm attaching one good run (Run 1 attachment) done previously with the same panel and protocol. The only difference is this time I stained 170Er CTLA-4 intracellularly and previously was surface stain. I didn't think it would make a difference and hence the antibody was not retitrated for intracellular staining. During the run, I noticed high streaking in the Ir191 and 193 DNA channels but all other channels had a reasonably low background. I am not sure if that is contributing to the high intensity of the negative population.

It would great to get your thoughts and if there is a way to salvage the experiment since I still have leftover stained sample (in 1.6% pfa in pbs) that could be acquired again.

Thank you!
Mansi
Attachments
cytof_12232020.pptx.zip
(624.92 KiB) Downloaded 348 times
<<

dgeanon01

Participant

Posts: 3

Joined: Fri Jan 24, 2020 6:03 pm

Post Sat Jan 09, 2021 7:32 pm

Re: Low resolution signal

Hi Mansi,

There are many variables that could be causing high background in your experiment, but my best guess is the following:

Many other users on this forum have reported higher signal background for surface markers when performing intracellular staining. Fixation is critical for CyTOF experiments to adhere both the antibodies and metal labeled polymers to their target cells, and the commercial fixatives used for intracellular staining notoriously degrade over time. Unless the fix/perm reagent used is relatively new (<1mo old), these reagents don't do a great job of fixing antibodies/polymers to target cells and therefore result in higher background staining across many markers. This could explain why staining quality was so great in your first experiment, but not in your second, as you included a fix/perm step post-surface stain in the second run.

To get around this obstacle, we routinely fix our cells (4%PFA, 10 minutes RT) post surface staining, prior to any fix/perm step (this preserves surface staining integrity in the context of fix/perm).

Unfortunately, this would imply that there is no way to salvage the staining quality of the cells from this specific experiment, as it's not possible to undo the damage done from the initial fix/perm.

Just a suggestion of what may have happened, we'd have to consider other possibilities if you did indeed do this initial cell fixation prior to fix/perm!

-Daniel
<<

narulam

Participant

Posts: 9

Joined: Sun Mar 29, 2020 10:28 am

Post Mon Jan 11, 2021 2:09 am

Re: Low resolution signal

Hi Daniel,

Thanks for your suggestions! I always stain some markers intracellularly, the only difference is ctla-4 is surface stained usually while it is stained intracellularly this time (though I don't believe that would cause high background). Both runs were in fact fixed with 1.6% PFA for 10' post surface stain and before intracellular staining (that is the only fix step in my protocol). So there is no change in the protocol. Two things that come to mind are insufficient washing steps that might be causing non-specific staining and the fresh bottle of PFA that I opened was bought 2-3 years ago that might have compromised the effectiveness of fixation.

I couldn't salvage the run like you said but will try to troubleshoot with better washing and testing newer PFA. Thanks a lot!

Mansi
<<

dgeanon01

Participant

Posts: 3

Joined: Fri Jan 24, 2020 6:03 pm

Post Tue Jan 12, 2021 9:34 pm

Re: Low resolution signal

To confirm, the surface markers (those you shared in your slides) were stained surface, correct? Or were they stained after permeabilization along with CTLA-4?
<<

narulam

Participant

Posts: 9

Joined: Sun Mar 29, 2020 10:28 am

Post Tue Jan 12, 2021 10:12 pm

Re: Low resolution signal

The markers in the slide were surface stained. Other markers such as foxp3,ctla4 (run2 only), RORgt, etc were intracellularly stained and are not shown in the slide.Thanks!
<<

narulam

Participant

Posts: 9

Joined: Sun Mar 29, 2020 10:28 am

Post Wed Jan 13, 2021 11:09 pm

Re: Low resolution signal

Hi,

Just to update everyone, pfa was not the problem, increasing the number of washes and absence of streaking in the Ir-DNA channel was able to fix the issue.

Return to CyTOF troubleshooting

Who is online

Users browsing this forum: No registered users and 3 guests

cron