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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.
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[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.
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JING LOU JinZhou medical university
HI, I have a qestion, I always got less than 105 cell on Flow cytometry detection ，iSn't it differentiation?
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Jia Li (Author)Department of Immunology, Medical Center,Duke University
May I know which population you detect is less than 105? What's the number of cells when you start this experiment? The latest modification time: 10/23/2016 4:52:54 PM
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.
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Menglan ChengInstitute of Virology, CAS, China
Are you sure about the concentration of neutralizing antibody, ng/ml or ug/ml?
Hassan HassanInstitute of Medicine
Hi!Are you sure about IL-2 in the Th17 polarizing medium? Often ANTI IL-2 is used as a neutralizing antibody, to decrease the risk of Th17 cell differentiation becoming inhibited.
Muzamil WantTHSTI
Hi, I would like to know that for how many days plate coated with antCD 3 and antiCD 28 is stable at 4 C before use? Thanks Muzamil
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 latest modification time: 7/19/2015 9:58:28 AM
Dong-Ming SuUNTHSC, Cell Biology & Immunology
Reading your protocol with great interest. Since we will conduct this experiment, I have a question: It looks that you do not need APCs (antigen presenting cells) in your culture system, why? What is difference with or without APCs? Thanks!
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.
Vivian PingHarbin Medical University
Thank you for your attention.I need to know a protocol about human T-cell in vitro differention,such as th1,th2,th17,treg.It would be of great hornor for your help.感谢您在百忙之中回复。我想了解人的T细胞极化，是否有这方面的protocol呢。
Sorry, I did not work with human cells. You'd better to consult someone expert with human T cell differentiation.
Bio-protocol team Bio-protocol teambio-protocol
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.
John LingMD Anderson Cancer Center
Hi guys:A quick question for people in TH17 business: Do you need to select naive T-cell subset to start with for the T-cells differentiation/activation then to check the TH17 situation by measuring IL-17? or to start with pan T-cell population? Biolegend tech support saying that they start with pan T-cells. But some others told me that i need to start with naive T-cells. Which one is correct?Thx.John
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.
qian fansouthern medical university
Thank you for your sharing.My English is poor,so please ignore it.Actually I am planing to do this assay recently。However，PMA from Sigma-Aldrich is not available in my county now，so I am confused about whic another brand should i choose to buy this reagent。Can you recommend some for me!Thanks a lot!fan
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！！
Keshav KumarUW
Hi,I apologize for my 2nd question. Please ignore it.Could you please what % of CD4+ IFN-G+, CD4+ IL-4+, CD4+ TH17+ I should get at the end of the expt (5 days)?Please let me know. Thanks a lot in advance!KK
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,I am planning to do this assay for my research work. Could you please clarify my following questions?1] Do I need to wash the plate bound CD3 before I seed the cells? I am confused with the step no 5. Why do I need to change the cells to new plate on day 2? Do I need to wash the cells before I move the cells to the resting media from day 2?2] Does step 7 require 1 hr or 4 hr stimulation because I am not seeing enough IFN-G with 1 hr? Please let me know.Thanks a lot,KK
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."
Thomas DohlmanMEEI
Hello,Thank you for this helpful protocol. In step 6, should the new media contain anti-CD28?Thank you!
No need to add anti-CD28.
Fanglian HeDepartment of Biology, University of Pennsylvania, USA
Interestingly, there is a parallel discussion of this protocol on ResearchGate: http://www.researchgate.net/post/Naive_T_cell_from_mouse_does_not_go_through_in_vitro_th1_th2_any_suggestion--Fanglian
你好，能不能提供文献出处呢？
Bio-protocol Editorial Team bio-protocol.org
Per authors' request, relevant references have been added to References section of the protocol. Sincerely,Bio-protocol editing team
Louie kwangmount sinai hospital
I used this protocol, however my cells did not go through differentiation. after 7 days, I barely see less 1% INFg in Th1 and IL4 in Th2. any suggestion.
I think maybe your intracellular staining does not work
Tamer MansourMichigan state university
I am using miltenyi biotec for isolation of CD4CD25 Treg cells. The left over population should be CD4+CD25low lymphocytes. Can I use this directly for in-vitro differentiation? My question is other words is how much the purity of naive T cells is important for this experiment?Thank you in advanceTamer
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
How many CD4+CD44loCD62Lhi T cells could you sort from one mouse (WT)? How many wells did you use for each condition (Th0/Th1/Th2/Th17)? Many thanks.
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.
After you transfer the culture to a new plate, do you still need to have anti-CD3 and antiCD28?
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.
lin zhanglzu
您好，请问：1.步骤5，为何要换新板？新板需要预先包被anti-CD3吗？2.步骤6，说是只加中和抗体不加细胞因子？我没过这样的3.这样刺激一轮的话阳性细胞的比例有多少？谢谢您了！
您好，1，步骤5，因为anti-CD3的抗体会造成细胞死亡，所以转移到新板这一步是必须的，这也是为什么一开始anti-CD3需要plate-bound的原因，方便转板。如果用soluble anti-CD3的话就需要洗细胞。2，步骤5，需要细胞因子。不好意思，已请编辑修改。3，对WT control来说，Th0的话看不到IL-4和IL-17，有10-15%的IFN-gamma，Th1的大部分细胞都表达IFN-gamma，Th2和Th17有15-20%左右的阳性率。
The step 5 has been updated.