Hi, while I don't have a lot of experience with T cells in particular, it is not uncommon to have your population of lymphocytes die upon stimulation, as lymphocytes are extremely sensitive to many death signals and are "programed" to die after blasting as a normal part of the contraction phase of the response. In addition, your media and the especially the pH of the media is crucial for cell survival. To keep your cells happy you may want to carefully track the pH of your media throughout the experiment.  From what I remember, your media should be pinkish in color. Yellow color indicates that your pH is off or that your wells are overgrown with cells (or both). Best of luck!

Your comments correlate with over-stimulation, quick proliferation and high cell activity that lead to apoptosis/necrosis. You should dilute or modifed the stimulation cocktail    composition and/or concentrations to tune it properly to your cells behaviour.

I think it is a problem with too strong stimulation of your cell during expansion and a too short resting period. Please have a look at Krug et al. In CII 2014 or 2015. It describes a GMP expansion protocol for human T cells without magnetic sorting. Whenyou scale that down, these cells should work. 

Good luck!

For the human CD3+ T cells, I often culture it in the presence of 10-20U IL-2. For stimulation, I use 1ug/ml PHA instead of PMA and iono. I think the in vitro culture period of health T cells may last within a week. If u want to do intracellular staining, culture T cells with stimulaory cocktail or pha, in the presence of BFA for less than 5 hours is enough. Good luck!

Jingying

If you only stimulate with anti-CD3/28 for 3 days follow by PMA/ION stimulation again. That would damage T cell whether caused by AID is a argument. Normally 7-10 days is ok before PMA/ION. Just check normal procedure for access T cell function (depends on what you look at). Yellowish medium is fine and change half medium is the best for T cell healthy.

Hi, Check the cells are ok and not exhausted before stimulation. Ideally, keep the cells in a concentration between 5 hundred thousand to 1 million cells per mL maximum. Do not wait for the media to become yellow split them before it happens. Cytotoxic T cells are extremely metabolic active and they pratically double their number daily. Do not stimulate a huge number of cells per well (in a small volume), because PMA is already a strong stimulation for them. Check the concentration of PMA. I have already done 4 hours stimulation and the time was ok for me. Good luck!

Hi Florian-

You may also need to titrate the anti-CD3/CD28 coated on the plates, and/or use soluble CD28 with plate-bound CD3.  Additionally, there are different antibody clones with different simulatory capacities-perhaps look into this and test different combinations. Also, check Miltenyi Biotec's site, they have good suggestions for well sizes, number of cells/well, and concentration of cells to use for in vitro stimulation protocols; as Marcos indicated, you have to be careful not to crowd the wells.  Further, what is the goal of your assay; ie., do you really need to do the initial stimulation or could you directly assess the cytokine profile via PMA/Iono stimulation?

• I would reduce your PMA to 5 ng/ml, maybe using 50 ng/ml is the cause of your excessive cytotoxicity, it does seems high to me. Good luck

I agree with most of the comments here. 

- Don't let the cells overgrow and the medium turn too yellow, if that is the case, optimize your cell numbers/culture volume for a given stimulus.

-Make sure you have 2-beta ME in your medium, that improves T cell survival through allowing a more reductive environment for proper T cell metabolism during expansion (at least for mouse T cells).

- Use soluble anti-CD28 and coated anti-CD3. Eventually scale down and/or let the cells rest/expand longer before you hit them with PMA/Iono/Brefeldin-Monensin. It is quite normal to induce cell death when you add that last stimulus, but it definitely shouldn't drop down to 2%!  make sure you add fresh medium (replace the medium) when you add this last stimuli. You should have at least 30-50% survival.

-  Also, mouse and human T cells behave differently, as some people already stated here. I would suggest that you add that information to this thread to get the best advice. 

Good luck!

Dear Florian, I agree with previous comment, it seems you are overstimulating cells and they are dying for AICD.

It would be nice to understand which is the most critical point:

you overstimulated them to much since the beginning (CD3/CD28/IL2) or later (PMA/Iono)?

I think you can try one titration experiment where you set up several conditions:

2-3 doses of anti-cd3/CD28

2-3 doses of IL2

2-3 doses of PMA/iono

PMA/iono are potent stimulator, not sure that you need to couple them with other coktails!

I'm aware that at the end you'll have many conditions to analyze, but this may hel you to understand what is oging on. You could also try this titration experiment just with total PBMCs, as it will be much cheaper and quicker....

and then adapt the findings with Naive

Hey,

thanks for all the answers so far :) It's mouse T cells ... forgot to mention that. I use 2-ME in the medium.

As many of you stated already, for me it's also overstimulation for some reason ... right now I have an experiment running to track their growth and to determine, when they actually stop dividing and rest.

I will also do the stimulation of whole splenocytes, as I anyways plan to analyze them with flow I can easily track down the CTLs. Long term plan is to do RNASeq on CTLs though and for that I will need a pure culture and I'd prefer the in vitro expansion over FACS.

As often with cells, we probably just have to get to know each other better and then I hope it works out ... and thanks for all the suggestions, I will work them out!

Will keep you updated :)

I agree with everyone mentioning overstimulation. 100 U/ml IL2 are too many units according to my experience. Try to use it at 20-50 U/ml. And also try to decrease PMA-Iono concentration (half or even 4x).  Good luck!

As promised, here's an update.

My very first RG-question really paid off, as it looks like. I tried to incooporate the suggestions and did the following:

Seeded the cells with a proliferation dye on CD3/CD28 coated 96-wells for two days. After two days splitted them 1:2 on uncoated wells. Followed proliferation and got a really nice peak-pattern, which diluted over days. On day five (the original day of restimulation of my old protocol), I decided to split them again as I still saw a huge difference to the pervious measurement in proliferation. I kinda concluded if they are still heavily proliferative, they might not really be resting. After another two days I saw, that the proliferation dye focuses as (almost) one peak, so I concluded that cells are resting now. I stimulated the cells with PMA/Ionomycin (50/500 ng/ml and 1:10 diluted) and other two wells with the 500x Stimulation Cocktail and 1:10 diluted. I measured viability and IFNg intracellularly. PMA/Ionomycin gave nice IFNg+ cells, 1:10 diluted a lot less positive cells and the viability was generally between 60-70%. As opposed to 80% viability without stimulation. I kept the IL2 at 20 U/ml ... so, hoping for reproducibility, I conclude that I'm on a really good way now thanks to you.

THANKS!!!

Dear Florian,

I am currently optimizing my protocol to study T-cell proliferation on primary mouse T-cells. I also use anti-CD3/CD28 to stimulate them, but I don't split them after.. maybe I should consider that.

Also, which type of 96-well plate do you use? have you seen any difference in the T-cell stimulation when using round-bottom versus flat-bottom plates?

And last question: do you use CFSE dye to analyze proliferation? and do you analyze it with the FlowJo software? I am currently having troubles with that..

Thanks in advance!

Best,

Alba

Quick question, I do not know if you mentioned it but are you adding feeder cells?