CyTOForum

Hi Everyone,
I am having issues with single tube sample acquisition. My runs have been going perfectly (no clogging) but when I check the data in flowjo, I am seeing some unusual bkg. I have discussed this with Fluidigm and they cannot seem to figure out where it is coming from. My staining protocol involves Cisplatin for LD, Surface markers, Fox p3 transcription buffer fix/perm, intracellular/intranuclear staining, and finally 2%PFA+Ir overnight. The bkg is not in every sample and there is nothing unusual that took place with the sample acquisition. From 18 samples/week I am seeing maybe 2-3 with signifcant Bkg. At one point we thought it was bead contamination. I ordered new beads and still see it. It was also suggested that residual acids from nitric or wash, so in many cases, I only used water between samples. I am attaching a ppt.

Thanks so much for your help
Diane
(Nadeau lab)

Hi Diane,

Is this your first time using the new cadmium isotopes for conjugation? I recently tested 112Cd and 113Cd for 3 of our standard antibodies (cPARP and CD235ab/CD61, respectively), and saw very high background, which made me give up on using the cadmium isotopes. I followed the modified conjugation protocol from Fluidigm to the letter using the MCP9 polymer. Even bought HRP Protector for storage of the MCP9-conjugated antibodies.

We typically use CD235ab/CD61 on 113In and cPARP on 161Dy with no problems. FYI, all of our antibodies are conjugated in-house.

PPT attached showing the difference. Hope that helps.

-Ed

Hi Ed and Diane,

Ed: I'm a bit confused by your PPT. Your Cd signal for the CD235/CD61 positive population is basically 1e4, which is outside the linear range of the instrument. Even your cPARP looks like it could be titered down quite a bit. These are especially if you're concerned about spillovers in the neighboring Cd113/Cd113 channels.


Diane: I'm also a little unclear on your PPT. What kind of background are you seeing, exactly? For example, are you saying that the 175Lu signal in the 3rd plot on the top of Slide 1 is background? Or are you talking about the diagonal smear in the third plot at the bottom on Slide 1?

Have you noticed any correlation between the weird backgrounds and what "type" of sample it is? For example, if I understand your PPT correctly, the Unstim is OK, but the Peanut (Stim?) is not. So, there's always the possibility that a background/contaminant signal is coming from your Stim reagents.

I would also say, the 114Cd and 116Cd metal solutions *will* have a certain amount of 113Cd signal in them. Remember, the CyTOF doesn't "know" the difference between In113 and Cd113....it only knows "mass 113". I don't *think* that's what's happening.


Side note: I would also strongly suggest that you Customize your Event Length Axis. Currently, you have it on asinh or some other exponential scaling. I would recommend that you switch that channel to Linear scaling and have it top out at 80 or 120 units; that will make your Ir vs EL gating much easier.


Mike

Hi Ed and Mike!

Thanks for your reply. It is the diagonal that I am referring to. I hope you were able to see the second ppt as well. It seems that this population is positive for 142Ce_beads. So for some reason, I am not able to exclude all the beads??? The samples are normalizing ok. Is there a problem with the IMD processing??

At first it did seem that it was only in samples with Cash, but I have since see them all populations (unstim, pea and cash).

I think that I can gate them out with CD4 and CD8, but if I have to analyze any population before this such as Live I will not get an accurate percent of positive cells.

Thanks
Diane

Hi Diane,

I was able to see both slides of the PPT (total of 2 slides). However, I only saw *one* PPT file; if there were more, I don't think they attached properly.

Could you post your *entire* staining panel (*all* reagents: livedead, Ir, Beads, Surface, Intracellular, etc.....)? It's not possible to look for potential spills without that information.

What event rate were you running these at? How streaky were things?


If you think there's a problem with IMD processing, then go back to the respective IMD and reprocess it. It's super rare for there to be a problem (and when it happens, it usually warps the entire file), but it does happen.


Mike

HI Mike,
Here is the gating and additional examples. I run my samples any where from 300-350. There was no smearing. The staining looked great.

Thank you

Hi Mike,
Here is my Panel. My staining is as following: After 4hrs BFA, Cisplatin, Surface, FoxP3 transcription fix/perm, perm wash, Intracellar/intranuclear staining, perm, CyFACS, 2%PFA+Ir O/N.

Thanks
Diane