r/Immunology

I am having troubles with activation of T cells. I use RPMI media supplemented with L-glutamine and add 10% FBS, 1% Pen strep, 1% NEAA, 25mM HEPES and on the day of isolation I add 50uM BME and around 40ng/ml of 200ug/ml IL2 (i didnt take note of the lot number so I dont know IU/ml :((() and 2ug/ml of CD28.

side note - i aliquot and store IL2 at -80. never use thawed vial again.

For activation, I precoat the plate with 3ug/ml of anti-CD3 for 2hours at 37C or o/v at 4C. I try to culture 1-1.5mln per ml in 12 well plate.

The main concern is I don't see them clustering/clumping together. They don't even float but feels like stuck on the plate bottom. even when i try to swirl, they are still kind of adhered to the bottom of the well. idk if it is a problem.

I believe I see them get bigger in size but just don't proliferate much.

And when I try to further chronically stimulate them, I see a lot of death on day 3-4 (2 days of activation + 1-2 days of further stimulation). so flow viability is like 7% (i know it is terrible)

anyone has any idea whats wrong with this flow? please help.......

Wrapping up my PhD in the next few months (🤜🪵), with a focus largely on neutrophils/NETosis. I've gotten a commemorative tattoo for each major lab I've been in, drawing on inspiration from tattoos other folks have gotten and shared.

That being said, I'm having a hell of a time finding any tattoos of neutrophils and/or NETs on any medium that I've searched. Anything I've found is completely textbook (no artistic flair) or AI slop, so I figured I'd crowd source to my fellow nerds! Anyone here with a neutrophil tattoo willing to share?

Hello, I work extensively with ENCODE data (if you're not familiar with this, it's a big consortium that produces and analyzes various aspects of human and mouse genomes and genetic regulation, broadly speaking), and I am curious about how some of the T cell samples I work with were activated, since it doesn't exactly align with the canonical three-part activation I learned about in my undergraduate immunology class.

Basically, I am looking at differences in genome organization between resting and activated T cells, but the treatment protocols continue to confuse me. Here is the description from the lab that did the actual activation:

"CD4+ and CD8+ T cells and their subsets were left resting in culture or activated using anti-CD3 and anti-CD28 (aCD3/aCD28) coated beads (Invitrogen cat# 11132D) (1 bead:2 cells ratio) and cultured in complete RPMI 1640 at 10M/6ml/well in 6 well-plate. Resting cells and 90% of activated T cells were collected on day 2 (36 hours) and beads were removed using Dynal magnet. The rest of activated cells was expanded in rhIL-2 (10 ng/ml, R&D Systems cat# 10453-IL-100) containing media for 5 to 6 more days and collected on day 7 or day 8 post activation. Activation of T cells was confirmed by the upregulation of CD25 and CD69 surface expression analyzed by flow cytometry. For differentiation into different T helper (Th) subsets, Naïve CD4+ (sorted CD45RO- CCR7+) T cells were activated with anti-CD3 and anti-CD28 beads in the presence of the following polarizing cytokine and blocking antibody combinations: Th0, medium only, Th1, human IL-12 (R&D Systems cat# 219-IL-025; 30 ng/ml) and neutralizing anti-IL-4 Antibody (R&D Systems cat# AF-204-NA; 1 μg/ml), and Th2, human IL-4 (R&D Systems cat# 204-IL-020; 100 ng/ml) and neutralizing anti-IFN-γ Antibody (R&D Systems cat# MAB285-100; 5 μg/ml). Cells were cultured and expanded in rhIL-2 (10 ng/ml, R&D Systems cat# 10453-IL-100) containing media for 2 weeks. On day 13 after activation, some of the cells were reactivated with aCD3/aCD28 beads (at 1 bead:3 cells ratio) for another 24 hours."

Can anyone tell me if this is standard procedure for in vitro T cell stimulation that closely mimics in vivo activation, or is there something off here that I need to be aware of?

Thanks in advance for reading this and for your help!!

Hi all!

I'm a biology graduate who loves drawing, so I've started making cartoons that explain immunology to people with no background.

My first one, "Picky Eater", shows a dendritic cell trying to feed a very fussy T cell: the watermelon has to be sliced (peptides), served on the right plate (MHC), and come with a fork (co-stimulation).

I've also written a plain-language article explaining the real science behind it.

I'd love some feedback from people, and ideas for what to cartoon next are welcome!!

Just started my PhD and I've already met 9 immunology couples. Is it proximity, shared trauma, or what? Cuz i have been definitely around other biological fields but never saw this drastic interpersonal interactions i see with immunology scientists

Hi, i was wondering if anyone here is familiar with mast cell biology. i have been doing immunophenotyping in muscle and lungs of balb/c mice. For some reason, the CD117+ Siglec F- cells that i have assumed are mast cells are not expressing Fcer1. I have done this with both control steady state mice and virus infected mice. However, my basophils are positive for the FceR1 receptor so i know the antibody works. i have seen this every time i've used this panel. i'm confused because im under the impression that mature mast cells in tissues should express FceR1.