Hi, I am trying to do in-vitro T cell differentiation from mouse naive CD4+CD62L+ T cells but there is something wrong that I cant figure out. I tried several times with different plates and antibodies but cells didn't proliferate at all. I coated the U-bottom nunc 96 well plate with 2ug/ml biolegend antiCD3e antibody (I tried with overnight 4 degree, 37degree or 2 hours 37 degree) and then I resuspended the cells in differentiating media that I prepared for Th1,Th2,Treg,Th17,Th22 with different antibodies. I am using complete media with 55mM b-merkaptoethanol, anti-CD28 concentration is 0.5 to 2 ug/ml. What could be the reason for cells are not proliferating or dying?
I used to do differentiations during my doctoral work. You can find my protocol below. You would notice that I used to use soluble a-CD3/CD28. You could coat the plates with a-CD3/CD28 or just a-CD3 and then use soluble a-CD28. We have noticed that using 10% serum (instead of 5%) makes a lot of difference. If your differentiation works, you should see good numbers of large cell clusters in your wells.
Lymph nodes and spleens are obtained from mice. The tissues are ground and the cells are then treated with an RBC lysis buffer to remove the red blood cells. The cells are then passed through a cell strainer to obtain a single cell suspension. The naïve CD4+CD62L+ cells obtained from MACS are resuspended in RPMI supplemented with 10% FCS, penicillin / streptomycin, glutamine, HEPES and sodium pyruvate.
Differentiation is done on 24 well plates. 0.5-1 X 106 naïve T cells are plated out per well. The cells are then stimulated with 5µg/ ml anti CD3 and 1µg/ ml anti CD28 in the presence of cytokines. The cytokine concentrations used for are as follows: Th17: TGFβ- 2ng/ml, IL-6- 30 ng/ ml, IL-23- 15 ng/ ml, IL-1β- 10 ng/ml, aIFNg- 1µg/ ml; Th1: IL-2- 2 ng/ ml, IL-12- 10 ng/ml, aIL-4- 1µg/ ml. The cells are cultured for 5 days.
On day 5, the cells are replated in restimulation media (RPMI supplemented with 10%FCS, penicillin/streptomycin, glutamine) and restimulated with ionomycin (750ng/ml) and phorbol- myristateacetate (50ng/ml) for 4 hours in the presence or absence of Brefeldin-A (5 μg/ ml). The cells restimulated in the presence of Brefeldin-A are used for intracellular cytokine staining.
Thanks for the answer Julie Joseph ! I guess my problem is about coating, I am coating the plate overnight with a-CD3 in PBS. what do you mean by %10 serum?
No worries. As I mentioned earlier, people coat differently. It is not incorrect to coat your plate overnight with a-CD3 in PBS. I meant have 10% serum in your culture medium. Cells seem to like it better. It could be a combination of factors. Try using the protocol I pasted above and hopefully it works for you.
Hi Sena nur Arbag
hopefully your cultures are doing fine by now. I just noticed that the b-Mercapto concentration seems too high. I think it should by µM and not mM.
Best regards
Robert
Divya Lairikyengbam , I have done human T cell culture but not polarization and so I would stick with what is in the literature. If I had to choose, I would do 6 days instead of 4. For restimulation, you remove the polarization media containing cytokines + antibodies and replace with media containing PMA + ionomycin. Again, maybe find a protocol for human cells and follow what is recommended.
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