Increase the time period of stimulation. Cells will start proliferating only after 48hrs

It seems really strange, i'm currently using the same beads and works nicely (at concentration of 25ul/10^6 cells).

After 48h PI staining shown a strong proliferation rate (around 50%, look at the attached file) and a bit of phase S / G2M is also visible 24h after activation, what protocol did you use for CD4+ T-cells purification from PBMC?

The cells could also show a delay in proliferation if you  hold it at 4°C before activation, the best choice would be activate them immediately after the purification.

How did you isolate your CD4+ cells from PBMC? Did you do a positive selection or a negative selection?

Thanks, all.  Luigi, your data looks great, is it 48 hrs results? Your beads/cell ratio is 1:1, right? And did you add IL-2? I use negative selection (EasySep™ Human CD4+ T Cell Isolation Kit, Stemcell), and activate it right after isolation. However, before isolation, the PBMC was put on room temperature for roughly 1 h 'cause I need wait for the centrifuge for another batch of blood, does it affect?

Negative selection is good and keep your cells in ice resuspended in media if you've to wait for long. Incubate for 72 h. before you check for proliferation. You should see some cell clumping. This should surely work, especially with IL-2 in the media.  

And use a viability dye to make sure your cells are actually alive, "1h in room temp"might be the problem.

Thanks, Sudipta. I just want to see the activation, so I didn't add IL-2. And I check cell cycle by flow, 60% cells are alive, 20% are debris, and another 20% are aggregate, is it normal?

Yes, the data are taken at 48 hours and the ratio is 1:1 like yours, i usually do not add Il-2 to not influence the system, but, for shure, with Il-2 you can improve the proliferation rate. 

I'm agree with Sudipta that the problem may have been the time RT (furthermore 60% of viability is too low), if everything goes well you can see cell clumpings like in the photo (or more) at 48h (20%aggregates is ok), the clumpings are already visible 24h after activation.

Thanks, Luigi. So can I put PMBC in ice or 4 °C instead of RT if I need wait centrifuge for 1-2 hours?

Yes, it's better keep the cells in ice (or at 4°C) if is possible in medium (LDA) or in PBS 3%FCS, in this way you slow down the activation of 3-4h but preserve all cells from death. It's important for optimal activation and proliferation keep apoptosis at low levels (max 3-4% after 48h of stimulation).

Good luck for the next experiment!

Luigi is correct, keep the cells in ice suspended in media. And the picture tells it all. If the proliferation is less you may not see a lot of clump under the microscope, but you should be able to see a few. Use some viability dye to detect how many live cells you are actually starting with before proliferation. You can use trypan blue to see if almost 90% of you cells are alive or not before you plate those. You can also try to use a viability dye in your cytometry ab mix. Good luck.

Thanks, Luigi and Sudipta, I'll try it again recently as you said. Appreciate for your help!

Dear Ge,

the addition of IL-2 is essential for expansion of T cells by Dynabeads. For the activation state you need to use 1 part of the protocol, but for the further transition between G1-S-M phases you need 2nd part (expansion). See, please, https://www.thermofisher.com/ru/ru/home/references/protocols/proteins-expression-isolation-and-analysis/t-cell-activation-and-expansion/dynabeads-human-t-activator-cd3-cd28.html

Hi Alexey,

Thanks for your information. I do use the protocol of part 1 from the link you provide. But it doesn't give out the details of experiments as we discussed here. 

Thanks.

Ge

I also failed to perform this experiment. I used positive selection T cell. I am afraid that that is the big problem for T cell activation. Can you share me more experiences?

Thank you so much.