u/EquivalentEqual7315

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.......

I am trying to see how different conditions change mitochondrial network in exhausted T cells. I will stain them with mitochondrial marker and then use imagej to look at the network. The problem is T cells are round and they have big nucleus and small cytoplasm. I dont think I will be able to see much.

I would appreciate if you shared your tricks and tips regarding the process!!!

Not sure if I provided enough details but I am happy to add more.

During my undergrad, I know I had to do clubs and organize different events and etc. however, I will be applying for jobs as a professional in my field. Do I still need to be part of grad program council and etc? Are there any other extracurriculars that are more fun than organizing happy hours?🥲🥲

Hi everyone!

I have been working on using dox-inducible CRISPR activation system to overexpress couple of genes of interest. I use 183411 and 183409 (addgene) plasmids, where I clone oligos into 183411 and co-transfect with 183409 plasmid that has dcas9 fused to transcriptional activators.

So, mostly the success should depend on oligos and I generated oligos using CHOPCHOP, crispick, and UCSC browser. However, whenever I treat transfected cells with dox, there is no change in band of interest but always random dox inducible band that is recognized by all antibodies of my proteins of interest and different sizes.

so the way system works is that my oligos serve as guides for dCas9-fused to activators and oligos guide it to the promoter site of the gene of interest, resulting in transcriptional activation and more expression.

Has anyone ever faced this issue? I have no idea how to troubleshoot it. There is def something being induced but I doubt that it is my protein. then what is it?

please help!!! (attached pictures of TWO western blots with TWO different proteins of interest but SIMILAR UNspecific dox inducible bands that also seem to be dox concentration-dependent (one seems to be around 100kd and the other is 70kd)

western 1 with POI A

western 2 with POI B

Hi everyone!

I am using double nickase plasmid from santa cruz to knock out a gene. After transfection with the plasmids, I am selecting with puro for a day and sorting gfp positive cells into 96 well plate to grow single colonies.

Then I want to introduce another gene but available plasmids have only puro selection or gfp.

I was wondering if my KO will be puro resistant or gfp-positive. And if it is absolutely bad idea to try to introduce another gene.

I feel like double nickase plasmids are more transient and won't be stably integrated?