Dear Yinlei Bai

I briefly wanted to reach out concerning your question.

First tip is to check, which anti-CD3 clone you have used as there are certainly differences between different clones available.

Here's a paper that lays that out nicely, but for stimulation of human T cells.

https://www.jimmunol.org/content/165/11/6205

The second tip is to add some IL-2 into your cultures, if that does not interfere with your experimental setup.

If you used the strongest activating antibody and IL-2 is not an option, you could switch to increasing the cell density, when seeding your cultures, which sometimes improves the viability of sorted cells. Moreover, the addition of non-essential amino acids and (expensive) Hyclone FBS, may in general improve the viability of your cultures.

Good luck with your experiments!

All the best,

Michael

Agree with what Michael said, check the anti-CD3 mAb clone for sure, and those anti-CD3 concentrations are on the high end, generally 1 ug/ml for coating suffices, especially with additional soluble anti-CD28. Also, the stronger anti-TCR signal without co-stimulatory signals, the more cell death you will obtain. What is your purpose with this experiment though? If you tell us that, we can give you better advice. You can definitely add some IL-2 to help them survive and proliferate. Keeping them close together and not sparse (96-well round bottom plates) also helps due to autocrine cytokines. It is very strange though that when you cultured total CD4+ the results were so different. Double check the purity of your starting populations by flow was well.

Hi, Yinlei Bai ..

What is the status of the remaining 10% live population on flow cytometry? What did you measure?

Michael Förster

Hi, thanks very much for your reply and suggestions. The anti-CD3 clone I used is 17A2 (bioXcell, cat#: BE0002). I don’t kwon whether it is strongest, but my lab usually use it with anti-CD28, both at 5ug/ml, to culture T cells and differention and everything is fine. About the cell density, how many cells would you seed?

Hi, Damian Maseda

thanks a lot for your suggestions. The purpose of my experiment is to test the effect of a co-inhibitory checkpoint on the proliferation of CD4 T cells, hence anti-CD28 (co- stimulatory) and IL-2 was not added. Can I use round-bottom plates to coat protein, e.g. anti-CD3? Thanks.

Hi, Amarendra Varma

I measured cell proliferation using CFSE method. The live cells proliferated.

Dear Yinlei Bai

I did a bit of searching and this is what I have found. I used 5 ug/ml plate bound anti-CD3 antibody to stimulated my murine T cells, as laid out here:

Article A role for the histone H2A deubiquitinase MYSM1 in maintenan...

The clone was 145-2C11, which does bind CD3epsilon and interferes with binding of 17A2. Both clones are comparative in their affinity, generally considered of medium and are therefore almost always used plate bound. You find a side by side comparison here:

https://academic.oup.com/intimm/article/20/10/1247/699489

Concerning the cell density, I would start at 5*105 cells per well in a flat or round bottom 96 well plate.

Here you find an in-depth discussion with references concerning Hyclone FBS, which made an enormous difference in my T cell cultures. So maybe it's worth a try.

Article Fetal Bovine Serum

All the best & kind regards,

Michael

Hi again Yinlei,

Yes, of course you can use round-bottom wells for coating. Also, make sure that your CFSe staining is not too harsh. I have seen often people overdoing it, and cells are very sensitive to this step: make sure you incubate a short time and that you wash and quench the remaining CFSE with washes with plenty of protein (like 10% FCS media). Good luck!

Hi, Damian, thanks for your answers. I have optimized CFSE labeling, usually 4min incubation is enough.

Hi, Michael Förster

Many thanks for your information. Is 5*10^5 cells too much? I seeded such many total CD4 T cells in a well of FORTY-EIGHT well plate coated by anti-CD3 and anti-CD28, and after D2-3, too dense.

Hi Yinlei Bai ...

It seems that the issue was not with your type of stimulation. Becuase, you said the live cells (~10%) proliferated as measured by CFSE staining.

This indicates that your stimulation of T cells with CD3 Ab alone worked well. Therefore, you do not need to worry about providing the co-stimulatory signal to your in vitro cultures.

I suggest you to wash your Ab coated wells carefully before seeding the cells. Check the colour of the medium periodically and split the cells or replace with fresh medium accordingly. Alternatively you can also observe the cells under the microscope to check how they look like or stain the cell sample with trypan blue when in doubt, before going to CFSE.

Amarendra Varma Hi, Thanks for yur suggestion!