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[Bio101] In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells
[Bio101] 小鼠原初 CD4 T 细胞到Th0, Th1, Th2, 和 Th17的体外分化方法   

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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.

Materials and Reagents

  1. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
  2. Penicillin/streptomycin solution (Life Technologies, Gibco®, catalog number: 15140-122 )
  3. 2-mercaptoethanol (Life Technologies, InvitrogenTM, catalog number: 21985-023 )
  4. IL-2 (R&D systems, catalog number: 402-ML-020 )
  5. IL-4 (Biolegend, catalog number: 574302 )
  6. IL-12 (R&D systems, catalog number: 419-ML-010 )
  7. Anti-CD3 (Biolegend, catalog number: 100302 )
  8. Anti-CD28 (Biolegend, catalog number: 102102 )
  9. Anti-IL-4 (Biolegend, catalog number: 504102 )
  10. Anti-IL-12 (Biolegend, catalog number: 505303 )
  11. Anti-IFNg (Biolegend, catalog number: 505702 )
  12. Phorbol 12-myristate-13-acetate (PMA) (Sigma-Aldrich, catalog number: 79346 )
  13. Ionomycin (Sigma-Aldrich, catalog number: 19657 )
  14. Monensin (ebioscience, catalog number: 00-4505-51 )
  15. Easysep CD4+ T cell enrichment Kit (STEMCELL Technologies, catalog number: 19752 )
  16. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-093 ) (see Recipes)

Equipment

  1. 48, 96-Well plate
  2. Centrifuges
  3. Water bath
  4. Fluorescence activated cell sortor (FACS)

Procedure

  1. Enrich CD4+ T cells by Easysep CD4+ T cell enrichment kit following the manufacturer’s instructions. Make sure the purity of CD4+ T cells is above 70% after enrichment.
  2. Sort CD4+ CD44loCD62Lhi naïve T cells from enriched CD4+ T cells.
  3. Count the sorted cells and plate them as follows:
    1. For 96-well-plate, 1 x 105 cells/well in 200 μl culture medium for each well.
    2. For 48-well-plate, 2 x 105 cells/well in 500 μl culture medium for each well.
  4. Set up the differentiation culture (numbers are final concentration)

    Th0
    Th1
    Th2
    Th17
    anti-CD3
    (plate coated)
    5 μg/μl
    5 μg/μl
    5 μg/μl
    5 μg/μl
    anti-CD28
    1 μg/ml
    1 μg/ml
    1 μg/ml
    1 μg/ml
    IL-2
    20 ng/ml
    20 ng/ml
    20 ng/ml
    20 ng/ml
    IL-4


    100 ng/ml

    IL-12

    20 ng/ml


    anti-IL-4

    10 ng/ml

    10 ng/μl
    anti-IFNg


    10 ng/ml
    10 ng/ml
    anti-IL-12


    10 ng/ml


  5. Transfer the culture to a new plate after 2 days without digesting or changing the medium.
  6. Culture for 3 more days. If medium turns yellow, replace it with new medium with neutralizing antibodies (no cytokines).
  7. Add 50 ng/ml PMA and 1 μg/ml ionomycin to the cells and incubate for 1 h.
  8. Add 2 μM monensin and incubate cells for another 4 h.
  9. Verify the lineage of T helper cells by determine the levels of intracellular IFN-gamma and IL-4 by FACS.
    Note: Please see the following link for common FACS staining protocol (http://www.bio-protocol.com/e31).

Recipes

  1. RPMI-1640 culture medium
    Supplemented with
    5% FBS
    1% Penicillin/streptomycin solution
    50 μM 2-mercaptoethanol

简介

在T辅助细胞研究中经常使用各种谱系的辅助T细胞的体外分化。 初始CD4T细胞可以在特异性刺激性细胞因子的存在下分化成T辅助细胞的某些谱系,并抑制对其他谱系分化必不可少的细胞因子。

材料和试剂

  1. 胎牛血清(FBS)(Gemini Bio-Products,目录号:900-108)
  2. 青霉素/链霉素溶液(Life Technologies,Gibco ,目录号:15140-122)
  3. 2-巯基乙醇(Life Technologies,Invitrogen TM ,目录号:21985-023)
  4. IL-2(R& D systems,目录号:402-ML-020)
  5. IL-4(Biolegend,目录号:574302)
  6. IL-12(R& D systems,目录号:419-ML-010)
  7. 抗CD3(Biolegend,目录号:100302)
  8. 抗CD28(Biolegend,目录号:102102)
  9. 抗IL-4(Biolegend,目录号:504102)
  10. 抗IL-12(Biolegend,目录号:505303)
  11. 抗IFNg(Biolegend,目录号:505702)
  12. 佛波醇12-豆蔻酸酯-13-乙酸酯(PMA)(Sigma-Aldrich,目录号:79346)
  13. 离子霉素(Sigma-Aldrich,目录号:19657)
  14. 莫能菌素(ebioscience,目录号:00-4505-51)
  15. Easysep CD4 + T细胞富集试剂盒(STEMCELL Technologies,目录号:19752)
  16. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-093)(参见Recipes)

设备

  1. 48,96孔板
  2. 离心机
  3. 水浴
  4. 荧光激活细胞分选机(FACS)

程序

  1. 通过Easysep CD4 + T细胞富集试剂盒按照制造商的说明书富集CD4 + T细胞。 确保CD4 + T细胞的纯度在富集后高于70%。
  2. 从富集的CD4 + T细胞分选CD4 + CD44loCD62L hi初始T细胞。
  3. 计数排序的单元格,并按如下方式进行排版:
    1. 对于96孔板,每孔200μl培养基中的1×10 5个细胞/孔。
    2. 对于48孔板,每孔500μl培养基中的2×10 5个细胞/孔。
  4. 设置分化培养(数字是最终浓度)

    Th0
    Th1
    Th2
    Th17
    抗CD3
    (镀板)
    5μg/μl
    5μg/μl
    5μg/μl
    5μg/μl
    抗CD28
    1μg/ml
    1μg/ml
    1μg/ml
    1μg/ml
    IL-2
    20 ng/ml
    20 ng/ml
    20 ng/ml
    20 ng/ml
    IL-4


    100 ng/ml

    IL-12

    20 ng/ml


    抗IL-4

    10 ng/ml

    10 ng /μl
    抗IFNg

    10 ng/ml
    10 ng/ml
    抗IL-12

    10 ng/ml


  5. 在2天后将培养物转移到新平板上,不消化或更换培养基
  6. 文化3天。 如果培养基变黄,请用新培养基更换为中和抗体(无细胞因子)
  7. 向细胞中加入50ng/ml PMA和1μg/ml离子霉素,并孵育1小时
  8. 加入2μM莫能菌素并将细胞再培养4小时
  9. 通过确定细胞内IFN-γ和IL-4的水平通过FACS验证T辅助细胞的谱系。
    注意:请参阅以下链接了解常见的FACS染色方案( http: //www.bio-protocol.com/e31 )。

食谱

  1. RPMI-1640培养基
    补充
    5%FBS
    1%青霉素/链霉素溶液 50μM2-巯基乙醇
  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, J. (2011). In vitro Differentiation of Mouse Th0, Th1 and Th2 from Naïve CD4 T Cells. Bio-protocol Bio101: e157. DOI: 10.21769/BioProtoc.157;
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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?
10/23/2016 8:50:49 AM Reply
Jia Li
SRI International

May I know which population you detect is less than 105? What's the number of cells when you start this experiment?

10/23/2016 4:52:21 PM


JING LOU
JinZhou medical university

1.5x105/well,

10/23/2016 11:04:25 PM


JING LOU
JinZhou medical university

th1,th17,treg , harvest three wells in each group,thanks!

10/23/2016 11:08:49 PM


Jia Li
SRI International

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.

10/23/2016 11:35:10 PM


Menglan Cheng
Institute of Virology, CAS, China
Are you sure about the concentration of neutralizing antibody, ng/ml or ug/ml?
8/2/2016 8:10:55 PM Reply
Hassan Hassan
Institute 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.
1/8/2016 7:29:26 AM Reply
Muzamil Want
THSTI
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
7/17/2015 12:10:15 AM Reply
Jia Li
SRI International

Coat with aCD3 one day before experiment and keep at 4 degree

7/17/2015 6:51:12 AM


Muzamil Want
THSTI

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.

7/19/2015 9:57:43 AM


Dong-Ming Su
UNTHSC, 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!
6/30/2015 10:33:19 AM Reply
Jia Li
SRI International

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.

6/30/2015 11:15:27 AM


Vivian Ping
Harbin 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.
3/9/2015 4:12:33 AM Reply
Jia Li
SRI International

Sorry, I did not work with human cells. You'd better to consult someone expert with human T cell differentiation.

3/9/2015 6:55:55 AM


Bio-protocol team Bio-protocol team
bio-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,

3/9/2015 4:13:51 PM


Vivian Ping
Harbin Medical University

Bio team,
Would you email me the protocol you mentioned.Here's my email,1411556509@qq.com.
Thank u.

3/10/2015 2:19:25 AM


Vivian Ping
Harbin Medical University

Jia,
thank you all the same.

3/10/2015 2:21:15 AM


John Ling
MD 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
2/9/2015 11:31:12 AM Reply
Jia Li
SRI International

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.

2/9/2015 12:12:29 PM


John Ling
MD Anderson Cancer Center

Thx a lot Jia for your comment.

2/10/2015 7:44:51 AM


qian fan
southern 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

11/26/2014 11:38:34 PM Reply
Jia Li
SRI International

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.

12/2/2014 8:27:44 PM


qian fan
southern medical university

Sorry for my late reply!And thank you very much!!

12/8/2014 6:28:42 PM


Keshav Kumar
UW
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
10/6/2014 10:36:16 AM Reply
Jia Li
SRI International

for WT CD4 T cells, there are a large fraction of INFg+ Th1, for IL-4 and IL17+, may be about 15-20%

10/12/2014 11:24:46 AM


Keshav Kumar
UW

Thanks a lot Jia!

10/12/2014 1:00:45 PM


Keshav Kumar
UW
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
10/6/2014 10:27:36 AM Reply
Jia Li
SRI International

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."

10/12/2014 11:21:23 AM


Thomas Dohlman
MEEI
Hello,

Thank you for this helpful protocol.
In step 6, should the new media contain anti-CD28?

Thank you!
10/21/2013 10:23:45 AM Reply
Jia Li
SRI International

No need to add anti-CD28.

10/25/2013 6:38:10 AM


Fanglian He
University of Pennsylvania
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
9/7/2013 12:27:38 AM Reply
8/27/2013 5:51:54 AM Reply
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

11/1/2013 12:51:54 AM


Louie kwang
mount 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.
8/7/2013 11:58:39 AM Reply
Jia Li
SRI International

I think maybe your intracellular staining does not work

8/7/2013 2:04:39 PM


Tamer Mansour
Michigan 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 advance

Tamer
7/8/2013 3:29:10 PM Reply
Jia Li
SRI International

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.

7/9/2013 1:40:35 PM


Tamer Mansour
Michigan state university

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!!

Regards

Tamer

7/22/2013 5:21:20 PM


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.
5/2/2012 4:59:35 PM Reply
Jia Li
SRI International

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.

7/9/2013 1:27:43 PM


After you transfer the culture to a new plate, do you still need to have anti-CD3 and antiCD28?
3/1/2012 7:33:21 AM Reply
Jia Li
SRI International

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.

3/2/2012 2:32:42 AM


lin zhang
lzu
2/17/2012 2:26:23 PM Reply
Jia Li
SRI International

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。

2/24/2012 3:00:41 AM


Fanglian He
University of Pennsylvania

The step 5 has been updated.

2/25/2012 3:37:56 PM