u/southernqueer96

ETA: TL;DR This is only happening with a handful of samples and seems to be an issue with the mastermix. I just don’t know what the issue could be.

Hey all, I’m back with more weird data. See: the weird diagonal streak that’s CD66b-super-low and CD19-negative-but-increasing, and the corresponding CD66b+CD19+ population. Panel, gating to this point, and compensation matrix (not edited; from Live/Dead on one of the non-weird samples) are in subsequent images.

These samples are fresh peripheral whole blood, each from a different human donor. All samples were drawn around the same time and stained within 2 hours of the first draw. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]

As indicated, the first four were stained with one mastermix and the last two were stained with a second mastermix (I thought I was getting four samples but then was told I’d be getting two more). Both mixes were made on the same morning within a couple hours of each other, and all of the samples were stained at the same time, so there should be no differences in processing aside from having two different mastermixes. They were all acquired on the cytometer at the same time and using the same compensation tubes.

I had seen the same streak with a few of my previous experiments, and I assumed it was a compensation issue, but based on these data, it seems like it might be a mastermix issue instead? What could possibly be causing that?

I pre-stained the samples with Fc block and monocyte block, and I used brilliant stain buffer for the mastermixes.

I tried applying comps from a different day to this data, and that didn’t make any difference.

It doesn’t seem to just be one marker - looking at the weird populations, they’re super high for some markers and negative for others -

CD66b-extra-low/CD19-increasing: ~70% CD8+, ~60% CD27+, ~70% CD14+, 100% HLA-DR+, ~40% CD127+, ~60% CD123+, 100% CD16hi, 100% CD3+, 100% CD57+, ~15% CD11c+, ~25% CD4+, 100% CD56+

CD66b+CD19+: All far left on live/dead gate, ~25% CD8+, 100% CD27%, mixed CD14 positivity, 100% HLA-DR+, 100% CD127+, mostly CD123-, 100% CD16 extremely high (~10E6), all CD3 very negative, all CD57 very negative, 100% CD11c+, mostly CD4-, 100% CD56+

In general, extremes for one marker correlate to extremes for the other markers.

For analysis, would you just gate higher on the CD66b- to cut that out? It’s about 3% of the PBMCs.

Thanks!

The three plots shown are peripheral whole blood samples from three different human donors. All samples were drawn around the same time and stained within 2.5 hours of the first draw, with a mastermix that was made the same day. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]

My gating strategy up to CD45 is shown in the second image.

I’m wondering if the CD45- SSC-Amid/hi population on the far right graph is just debris or something else? If it’s just debris, is it a processing issue or just biological?

This is not the only donor I’ve seen it in, just an example. I see it in 14/48 samples that I’ve analyzed for the same study. It doesn’t seem to correlate with the date (ie, which samples are stained and run at the same time), the time from draw to processing, or disease process/severity of illness.

I assume it’s fine to be gating it out, but I want to double-check.

I got this second-hand, so I have no idea where it was purchased. I don’t see any brand labeling on it (unless the marking above “E27” on the light is a brand logo). It’s worked well for me for ~5 years (don’t know how long it was used before that) but has stopped working. I bought a Sansi in a similar style based on recommendations, but I don’t like it nearly as much - the clip-on clamp doesn’t work as well, the necks are significantly shorter, and the lights get hot enough to burn myself. I’d love to buy another of my old light if I can find it. Thanks!

This was a few years ago, but I realized that I never asked for an ID at the time. Hope these pics are good enough! It was very angry to be trapped 😬 I did release outside after taking pics.

I’ve moved it outside in a jar, granted I’m not sure how healthy it is given how sluggish it seemed
:( When I opened the jar, it crawled out and circled the rim of the jar for a bit. I went back inside after a few minutes, and it hadn’t flown away yet. I realized after looking at these photos that it might be missing a couple of legs as well.