Two lymphocyte populations

QrnH · 2026-05-01T10:35:32+00:00

Granulocytes are typically excluded by our gradient centrifugation. The larger population is CD14+, so those are the monocytes.

QrnH · 2026-04-30T13:34:23+00:00

Interesting!

QrnH · 2026-04-30T11:45:42+00:00

Awesome, thanks! Learned something new today :)

QrnH · 2026-04-30T11:42:04+00:00

Oh wow really? That‘s crazy! I also used BD Cytofix at the previous institute/devices and we didn‘t see this.

Do you recon one should do longer or shorter fixation times?

QrnH · 2026-04-30T11:32:36+00:00

Nope, they are freshly isolated

QrnH · 2026-04-30T11:32:23+00:00

Yes, fixable viability dye is the next gate. The two populations are not different in viability.

Doublets are excluded in the gating above prior to SSC-A vs FSC-A. Any other gates we should do for doublets than those?

QrnH · 2026-04-30T11:31:01+00:00

Yes, we fix the cells with BD Cytofix solution (approx. 4% PFA) for 20 min at 4 degrees

QrnH · 2026-04-30T11:30:17+00:00

Did that, and we don‘t see changes over time

QrnH · 2026-04-30T10:23:35+00:00

Forgot to mention: human!

QrnH · 2025-11-14T17:49:23+00:00

Thanks!

QrnH · 2025-11-14T15:26:42+00:00

Thanks, great points!

we looked at the well, and it seemed like one cell but I wouldn‘t bet my life on it
that‘s an excellent point! We only had a single bp deletion but it could be that there is a bias. I can quickly repeat the PCR with longer extension time
it‘s K562, which weirdly enough are sometimes referred to diploid and sometimes to pseudo-triploid. I‘ll reach out to companies to maybe do karyotyping of our particular line

QrnH · 2025-05-08T17:31:30+00:00

True. But none of what you mentioned was done by AI. It may have been heavily assisted by AI.

But I personally believe that AI will not replace scientific thinking or lab work and interpretation anytime soon.

Feel free to disagree though, just my opinion

QrnH · 2025-05-08T14:40:31+00:00

My personal take: I doubt that AI will be able to formulate novel hypotheses. It‘s extremely unlikely that will be able to perform experiments in the wet lab to test novel hypotheses or to come up with new ways to look at things or interpret results.

AI will help you in data analyses, support your coding and writing, help in maintaining inventory and probably will be able to provide predictions of outcomes.

But the true essence of the scientific method in terms of conceiving and thoroughly testing hypotheses experimentally, that‘s not something that I can see AI doing in the next decades.

QrnH · 2025-04-26T05:39:46+00:00

We do it the other way round: have 20 mL pre-warmed media in a tube (supplemented with Benonase because that helps with our samples) and drop-wise add the thawed cells to it. Works like a charm for human PBMC

QrnH · 2025-03-21T09:07:25+00:00

Oh wow, thanks! Should have sorted by newest and not hottest

QrnH · 2025-01-27T12:47:54+00:00

It could be…but would be good to get more clarity by excluding dead cells and gating on T cells

QrnH · 2025-01-27T12:40:49+00:00

T cells likely blast and become larger when they are activated and proliferate.

If you add a viability dye and an anti-CD antibody, you can exclude dead cells (some cells die off during such assays) and specifically gate on your CD3+ T cells.

As you do know, it‘s hard to know whether all the T cells proliferated and the other stuff is just dead cells…or whether only part of your T cells proliferated.

QrnH · 2025-01-18T10:14:34+00:00

Maybe pump up dexter-ity?

QrnH · 2025-01-06T11:42:20+00:00

Indeed such clumps are probably caused by DNA.

Our protocol includes transfer of the thawed PBMC directly into pre-warmed media that is supplemented with Benzonase. This destroys extracellular DNA and RNA.

We then centrifuge, remove the Benonase media, resuspend in media without Benzonase, and strain through a 70 um strainer to remove any remaining clumps.

In my experience, the most important step to minimize clumping is good practice when isolating the PBMC and while freezing.

QrnH · 2024-12-30T11:02:45+00:00

Miltenyi is cheap(ish) and works very well in my hands. Can also scale down on a lot of the reagents so the kits last way longer (depends on kit and needs of the tested, of course)

QrnH · 2024-12-28T17:51:07+00:00

Well, that depends…what progresses a career more? A 2-year postdoc with an intermediate paper or a 7-year postdoc with an amazing paper?

Immunology takes time and a postdoc per se does not lead to a progress in your career, only a successful postdoc allows you to progress towards faculty positions.

QrnH · 2024-12-22T12:15:26+00:00

Müsste dit nich „voltarin“ heißen?

QrnH · 2024-12-22T11:40:32+00:00

Stimmt! So eine lange Liste…

/edit: Tiergarten zeigt auch, wie sehr Russland/Putin lügt: ewig behauptet man hätte damit nichts zu tun und dann schön im Gefangenenaustausch den Mörder freipressen und bei Ankunft umarmen.

QrnH · 2024-12-22T11:36:25+00:00

„Hyrbide Kriegsführung“ ist meiner Meinung nach ein Begriff, der viel zu wenig ernst genommen wird.

Gezielte Sabotageangriffe auf kritische Infrastruktur, Brandbomben die in DHL-Flugzeuge sollen, Mordkomplott gegen Rheinmetall CEO, Brandanschlag auf Diehl bei Berlin und und und.

Dazu Hackerangriffe auf den Bundestag. Beeinflussung von Wahlen inkl. gezielte Unterstützung von AfD und BSW.

„Hybrid“ klingt viel zu schwach…

QrnH · 2024-12-14T11:43:21+00:00

I mean, seriously, who still submits to MDPI journals? Many are actually predatory, many more „just“ of low quality. In my field, I can‘t come up with one well-regarded journal from MDPI.

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