Abstract
In vitro differentiation of helper T cells of various lineages is frequently used in T helper cell study. Naïve CD4 T cells can differentiate into certain lineage of T help cells in vitro in the presence of specific stimulatory cytokines and inhibition of cytokines that are essential for the differentiation of other lineages.
Materials and Reagents
Equipment
Procedure
Recipes
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Hi,it‘s 5ug/ml. Sorry for the typing mistake
Thanks Jia Li for valuable support
Sorry I haven't done Th9 differentiation before, so cannot answer this question.
thank Jia Li, thanks a lot😊
May I know which population you detect is less than 105? What's the number of cells when you start this experiment?
1.5x105/well,
th1,th17,treg , harvest three wells in each group,thanks!
if you can detect IFNgamma and IL-4, they should differentiate into Th1 and Th2. For less cell No. problem, one possibility is that you lose many cells during staining and washing. You can count cells before you collect and stain them, if they are not less than 105, you need to think about your staining process. If before staining you have small number of cells, do not use CD3 after transferring cells to new plate, because anti-CD3 will cause cell death, that's why anti-CD3 is plate-bound.
Coat with aCD3 one day before experiment and keep at 4 degree
thanks Jia. I always coat the plate one day before but due to some problem in sorting of cells next day i could not plate cells. Thats why i asked for how many days i can keep the coated plate at 4 C.
The differentiation do not need peptide presented by APCs, and anti-CD3/CD28 can activate T cells. If you want to check the differentiation of peptide-specific T cell, you may need APCs instead of anti-CD3/CD28.
Sorry, I did not work with human cells. You'd better to consult someone expert with human T cell differentiation.
Hi Vivian,Just let you know that we recently received a protocol about T cell polarization assays in mice (not human T cells, though), which is under review. If you are interested, we would keep you posted on the status of the MS.Thanks,
Bio team,Would you email me the protocol you mentioned.Here's my email,1411556509@qq.com.Thank u.
Jia,thank you all the same.
I haven't work with Pan T-cell, but naive T cell works well. Just check the publications about Th17 differentiation, choose the proper one for your project.
Thx a lot Jia for your comment.
Sorry I haven't use the product from other companies. But I think PMA is not so special reagent, the same reagent from a well-reputation company should work.
Sorry for my late reply!And thank you very much!!
for WT CD4 T cells, there are a large fraction of INFg+ Th1, for IL-4 and IL17+, may be about 15-20%
Thanks a lot Jia!
Hi,1. Yes, wash the extra CD3. check the regular protocol for CD3 plate coating.see my previous answer:"Because anti-CD3 will cause cell death, that's why anti-CD3 is plate-bound and we transfer culture to new plate."check the protocol step 5" Transfer the culture to new plate after 2 days without digesting or changing the medium."
No need to add anti-CD28.
I think maybe your intracellular staining does not work
Sorry I haven't use this technique before, but I think CD44 and CD62L test are still necessary for the naive phenotype. Although some resting memory cell express CD25, there are CD25- effector and memory cells. I did not use CD4+CD25- population as the naive T cells, so cannot answer the purity question.
I am using now your protocol however on IL4 staining I am getting a significant shift in my sample compared to unstained control but no separate distinction of a positive versus negative population. What I am doing now is just adding the antibody in the perm buffer. I am using 11B11 antibody from Biolegend (504104) for flowcytometric analysis.N.B. I am getting ~75% positive cells for INfg after Th1 differentiation!!RegardsTamer
about several million naive cells can be obtained from WT. Usually at least three wells for each condition so that the results can do statistic analysis.
No need to add anti-CD3 and anti-CD28. Because anti-CD3 will cause cell death, that's why anti-CD3 is plate-bound and we transfer culture to new plate.