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In vitro Regulatory T cells Differentiation From Naïve T Cells

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Cancer Research
Feb 2013



In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using tumor cells conditioned media from mouse naïve T cells and to identify them based on their specifics markers (Dalotto-Moreno et al., 2013).

Keywords: Regulatory T cell (调节性T细胞), Isolation (隔离), Breast cancer (乳腺癌), T cells (T细胞), Naive T cells (幼稚T细胞)

Materials and Reagents

  1. Splenocyte suspension
    1. Eight- to twelve-week old Balb/c mice strain
    2. RPMI 1640 (Life Technologies, Gibco®, catalog number: 22400-089 )
    3. Phosphate buffer saline (PBS) (see Recipes)
    4. Sterile red blood lysis buffer (ACK buffer) (see Recipes)

  2. Cell lines
    1. 4T1 cell line (ATCC)
      4T1 is a highly metastatic stage IV murine breast cancer cell line that lacks estrogen and progesterone nuclear receptors and that spontaneously metastasizes to lung, brain and bone.
    2. RPMI 1640
    3. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10438-026 )
    4. 100x Antibiotic-antimycotic (Life Technologies, InvitrogenTM, catalog number: 15240062 )

  3. Determination and purification of CD4+ Treg and naïve T cells
    1. Allophycocyanin (APC)-conjugated anti-CD4 antibody (clone GK1.5) (eBioscience, catalog number: 17-0041 )
    2. Alexa Fluor 488-conjugated anti-CD25 antibody (clone PC61.5) (eBioscience, catalog number: 53-0251 )
    3. Phycoerythrin (PE) -conjugated anti-CD62L antibody (clone MEL-14) (eBioscience, catalog number: 12-0621 )
    4. PE-conjugated anti-Foxp3 antibody (clone FJK-16s) (eBioscience, catalog number: 12-5773 )
    5. Fix/Perm buffer (eBioscience, catalog number: 00-5123 , 00-5223 )
    6. 10x Permeabilization Buffer (eBioscience, catalog number: 00-8333 )
    7. Dynal® Mouse CD4 Cell Negative Isolation Kit (Life Technologies, InvitrogenTM, catalog number: 114-15D )
    8. Heat-inactivated fetal bovine serum (FBS) (Life Technologies, Gibco®, catalog number: 10438-026)
    9. FACS buffer (see Recipes)
    10. Sorted cells collection medium (see Recipes)

  4. Differentiation of Treg in vitro
    1. NA/LE Hamster anti-mouse CD3ε monoclonal antibody (clone 145-2C11) (BD, catalog number: 553057 )
    2. NA/LE Hamster anti-mouse CD28 monoclonal antibody (clone 37.51) (BD, catalog number: 553294 )
    3. Antibiotic-antimycotic (Life Technologies, InvitrogenTM, catalog number: 15240062)
    4. RPMI 1640 supplemented with 50 µM β-mercaptoethanol and antibiotic-antimycotic (Life Technologies, InvitrogenTM, catalog number: 15240062)
    5. Recombinant hTGFβ1 (R&D Systems, catalog number: 100-B ) (see Recipes)
    6. Recombinant mIL-2 (R&D Systems, catalog number: 402-ML ) (see Recipes)


  1. One milliliter syringe (BD, catalog number: 309628 )
  2. Sterile scissors
  3. P60 petri dishes (Greiner Bio-One GmbH, catalog number: 628160 )
  4. Sterile 70-μm filter (BD, catalog number: 352350 )
  5. Syringe filter (0.22 μm) (Corning, catalog number: 431219 )
  6. FACSAria cell sorter
  7. FACSAria II (BD, catalog number: 642510 )
  8. Airstream Class II BSC (ESCO Corporation)
  9. 15 ml conical tubes (BD, catalog number: 352095 )
  10. 5 ml polystyrene round bottom tubes (BD, catalog number: 352052 )
  11. Twenty four well plates (Greiner Bio-One GmbH, catalog number: 662160 )
  12. Centrifuge 5810R (Eppendorf, catalog number: 5811 000,622 )
  13. Dynal MCP-L (Life Technologies, InvitrogenTM, catalog number: 120.21D )
  14. CO2 incubator


  1. Isolation of CD4+CD62L+ T naïve cells
    1. Prepare a single cell suspension from mouse spleens. Disrupt the spleen with the plunger of a 1 ml syringe against a 70-μm filter in a petri dish filled with 2 ml of RPMI.
    2. Centrifuge single cell suspensions in 15-ml conical tubes for 8 min at no more than 300 x g.
    3. Re-suspend the splenocytes with 5 ml of ACK buffer and incubate 5 min at RT. Dilute it with PBS and centrifuge for 8 min at no more than 300 x g. Re-suspend cell pellet in FACS buffer and count cell number. Normally, each spleen yields between 80-100 x 106 splenocytes.
    4. Purification of CD4+ T cells by negative selection using Dynal® Mouse CD4 Cell Negative Isolation Kit is thoroughly detailed in the protocol provided by manufacturer. Protocol yield is usually 20-25% of spleen cells (http://tools.invitrogen.com/content/sfs/manuals/dynabeads_untouched_ms_CD4_man.pdf).
    5. After CD4+ T cells isolation adjust the cell concentration by centrifugation (8 min at 300 x g) and dilution in FACS buffer to 4 x 107/ml and proceed to CD4 and CD62L staining.
    6. Use 0.2 μg of APC-conjugated anti-CD4 antibody and 0.3 μg of PE-conjugated anti-CD62L antibody per 200 μl of CD4+ T cells suspension. Incubate 30 min at 4 °C in the dark.
    7. Wash cells with FACS buffer, centrifuge for 8 min at no more than 300 x g and re-suspend cell pellet with FACS buffer at a concentration of 3 x 107/ml.
    8. Using a FACSAria cell sorter proceed to the selection and sorting of the CD4+CD62Lhigh population. Exclude cell doublets using FSC-H vs. FSC-W and SSC-H vs. SSC-W dot plots. The total percentage should be between 60-70% for Balb/c mice strain and 50-60% for C57Bl/6 mice strain. Flow rate is recommended to be adjusted around 1-3. Sort precision could be set to “yield”. One should expect around 10 x 106 and 7 x 106 of CD4 T naïve cells per Balb/c and C57Bl/6 spleen, respectively.

    9. Use 15 ml conical tubes to collect sorted population with 2.5 ml of collection medium. Prior to use, vortex tubes so that tube walls will be covered by a thin layer of fluid thus avoiding cell death when cells are deflected to the tube.
    10. Keep the sorted population on ice.

  2. Conditioned media from tumor cells
    1. This step can be performed at any time prior to the Treg differentiation protocol.
    2. Plate the chosen tumor cells in P60 dishes at 50% confluence with 2 ml of serum free media. Incubate for 18 h at 37 °C with 5% CO2 and then collect conditioned media. Filter with 0.22 μm syringe filter, aliquot into 200 µl samples and store at -70 °C.

  3. Anti-CD3 coating of 24-well plates
    1. Prepare a 5 μg/ml solution of anti-CD3ε from the stock of CD3ε antibody (1 mg/ml) in sterile PBS and vortex. For 24-well plates use 150 μl per well.
    2. Incubate at 37 °C in a humidified atmosphere for at least 2 h.
    3. Before use rinse wells with PBS and aspirate twice.

  4. Conversion of naïve T cells to Treg in the presence of tumor cells conditioned media
    1. The stimuli indispensable for Treg conversion are TGFβ1 and IL-2. To asses Gal-1 fine-tuning of Treg conversion frequency it is necessary to use a limiting concentration of the former. TGFβ1 limitation has shown to be more efficient at modulating Treg differentiation. Adjust naïve T cells concentration to 1 x 106/ml in serum free-RPMI supplemented with 1-2 ng/ml hTGFβ1, 100 U/ml mIL-2, 1 μg/ml CD28 mAb and antibiotic-antimycotic.
    2. Plate 1 ml of naïve T cell suspension per well in anti CD3-coated 24-well plates.
    3. Add conditioned media (CM) tumor cells. It is suggested to determine dose-dependent responses to the CM. Dilutions ranging from 1:10 to 1:100 are recommended.
    4. Incubate at 37 °C with 5% CO2 for 4 days. More days will only result in an increased cell death.
    5. Asses Treg frequency by flow cytometry after staining of CD4, CD25 and Foxp3.

  5. Treg staining
    1. Staining of CD4 and CD25 molecules are performed for 30 min in the dark at 4 °C. Per 2 x 106 cells use 0.03 μg of APC-conjugated CD4 antibody and 0.075 μg of AlexaFluor 488-conjugated CD25 in 100 μl of FACS buffer.
    2. Wash cells and centrifuge for 8 min at no more than 300 x g. Fix and permeabilize cells using Fix/Perm buffer in 100 μl for 30 min to 18 h in the dark at 4 °C.
    3. Wash cells with 1x Permeabilization Buffer. Foxp3 staining is performed in 100 μl 1x Permeabilization Buffer using 0.225 μg PE-conjugated Foxp3 antibody for 1 h at 4 °C in the dark.
    4. Wash cells with 1x Permeabilization Buffer, centrifuge for 10 min at 300 x g and re-suspend in FACS buffer.
    5. For flow cytometry analysis a two-laser cytometer must be used and 5 additional tubes containing the appropriate compensation samples should be considered. It is highly recommended to exclude cell doublets using FSC-H vs. FSC-W and SSC-H vs. SSC-W dot plots.


  1. Phosphate buffer saline (PBS)
    136 mM NaCl
    8.2 mM Na2HPO4
    1.5 mM KH2PO4
    2.7 mM KCl (pH 7.4)
  2. Sterile red blood lysis buffer (ACK buffer)
    150 mM NH4Cl
    10 mM KHCO3
    0.1 mM EDTA
    Resuspend in distilled H2O
    Filter sterilize (0.45 μm)
    Stored at 4 °C
  3. FACS buffer
    PBS with 0.1% BSA and 2 mM EDTA
  4. Sorted cells collection medium
    RPMI 1640 supplemented with 20% FBS
  5. Recombinant hTGFβ1
    Dissolved in phosphate buffer saline (PBS) (pH 7.4) to a working dilution 30 µg/ml Stored in aliquots at -70 °C
  6. Recombinant mIL-2
    Dissolved in PBS to a working dilution of 10 μg/ml
    Stored in aliquots at -70 °C
    Note: Avoid repeated freeze-thaw cycles as it may lead to loss of activity.


This protocol is based in the original work published in Dalotto-Moreno et al. (2013). This work was supported by grants from Agencia Nacional de Promoción Científica y Técnica Argentina (ANPCyT; PICT 2007-093 to M.S. and 2010-870 to G.A.R. and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; PIP 2010-2012 to M.S. and G.A.R.), Fundación Sales to G.A.R. The authors wish to express special thanks to María Rosa Morales for animal technical help.


  1. Curiel, T. J., Coukos, G., Zou, L., Alvarez, X., Cheng, P., Mottram, P., Evdemon-Hogan, M., Conejo-Garcia, J. R., Zhang, L., Burow, M., Zhu, Y., Wei, S., Kryczek, I., Daniel, B., Gordon, A., Myers, L., Lackner, A., Disis, M. L., Knutson, K. L., Chen, L. and Zou, W. (2004). Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10(9): 942-949.
  2. Dalotto-Moreno, T., Croci, D. O., Cerliani, J. P., Martinez-Allo, V. C., Dergan-Dylon, S., Mendez-Huergo, S. P., Stupirski, J. C., Mazal, D., Osinaga, E., Toscano, M. A., Sundblad, V., Rabinovich, G. A. and Salatino, M. (2013). Targeting galectin-1 overcomes breast cancer-associated immunosuppression and prevents metastatic disease. Cancer Res 73(3): 1107-1117.
  3. Sakaguchi, S. (2004). Naturally arising CD4+ regulatory t cells for immunologic self-tolerance and negative control of immune responses. Annu Rev Immunol 22: 531-562.
  4. Zou, W. (2006). Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 6(4): 295-307.


在过去的几年中,表达CD4,CD25和转录因子FoxP3的调节性T细胞(Treg)的一个子集已经作为T细胞耐受性和体内平衡的关键调节剂获得了相当大的关注(Sakaguchi,2004)。 这种T细胞群特别参与维持免疫自身耐受性和控制对外来抗原的异常免疫应答。 值得注意的是,调节性T细胞通过抑制T细胞介导的抗肿瘤免疫参与了肿瘤细胞逃避免疫应答(Curiel等人,2004; Zou,2006)。 促进这种抑制群体在肿瘤微环境中的分化的信号的研究已经成为一个中心问题。 在这里,我们描述了使用来自小鼠天然T细胞的肿瘤细胞条件培养基来体外鉴别Treg的详细方法,并且基于它们的特异性标记物来鉴定它们(Dalotto-Moreno等人, >,2013)。

关键字:调节性T细胞, 隔离, 乳腺癌, T细胞, 幼稚T细胞


  1. 脾细胞悬液
    1. 八 - 十二周龄Balb/c小鼠品系
    2. RPMI 1640(Life Technologies,Gibco ,目录号:22400-089)
    3. 磷酸盐缓冲盐水(PBS)(见配方)
    4. 无菌红血液裂解缓冲液(ACK缓冲液)(见配方)

  2. 细胞系
    1. 4T1细胞系(ATCC)
    2. RPMI 1640
    3. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10438-026)
    4. 100x抗生素 - 抗真菌剂(Life Technologies,Invitrogen TM ,目录号:15240062)

  3. CD4 + Treg和天然T细胞的测定和纯化
    1. 别藻蓝蛋白(APC)结合的抗CD4抗体(克隆GK1.5)(eBioscience,目录号:17-0041)
    2. Alexa Fluor 488-缀合的抗-CD25抗体(克隆PC61.5)(eBioscience,目录号:53-0251)
    3. 藻红蛋白(PE)缀合的抗CD62L抗体(克隆MEL-14)(eBioscience,目录号:12-0621)
    4. PE缀合的抗Foxp3抗体(克隆FJK-16s)(eBioscience,目录号:12-5773)
    5. Fix/Perm缓冲液(eBioscience,目录号:00-5123,00-5223)
    6. 10x渗透缓冲液(eBioscience,目录号:00-8333)
    7. Dynal 小鼠CD4细胞阴性分离试剂盒(Life Technologies,Invitrogen TM ,目录号:114-15D)
    8. 热灭活的胎牛血清(FBS)(Life Technologies,Gibco ,目录号:10438-026)
    9. FACS缓冲区(参见配方)
    10. 分选细胞收集培养基(参见配方)

  4. 体外分化Treg
    1. NA/LE仓鼠抗小鼠CD3ε单克隆抗体(克隆145-2C11)(BD,目录号:553057)
    2. NA/LE仓鼠抗小鼠CD28单克隆抗体(克隆37.51)(BD,目录号:553294)
    3. 抗生素 - 抗真菌剂(Life Technologies,Invitrogen TM ,目录号:15240062)
    4. 补充有50μMβ-巯基乙醇和抗生素 - 抗真菌剂(Life Technologies,Invitrogen TM,目录号:15240062)的RPMI 1640。
    5. 重组hTGFβ1(R& D Systems,目录号:100-B)(参见Recipes)
    6. 重组mIL-2(R& D Systems,目录号:402-ML)(参见Recipes)


  1. 1毫升注射器(BD,目录号:309628)
  2. 无菌剪刀
  3. P60培养皿(Greiner Bio-One GmbH,目录号:628160)
  4. 无菌70μm过滤器(BD,目录号:352350)
  5. 注射器过滤器(0.22μm)(Corning,目录号:431219)
  6. FACSAria细胞分选机
  7. FACSAria II(BD,目录号:642510)
  8. 气流II级BSC(ESCO Corporation)
  9. 15ml锥形管(BD,目录号:352095)
  10. 5ml聚苯乙烯圆底管(BD,目录号:352052)
  11. 二十四孔板(Greiner Bio-One GmbH,目录号:662160)
  12. 离心机5810R(Eppendorf,目录号:5811000,622)
  13. Dynal MCP-L(Life Technologies,Invitrogen TM,目录号:120.21D)
  14. CO <2>孵化器


  1. 分离CD4 + + CD62L +/+ T细胞
    1. 从小鼠脾制备单细胞悬液。用填充有2ml RPMI的培养皿中的1ml注射器的柱塞对着70-μm过滤器破坏脾脏。
    2. 将单细胞悬浮液在15-ml锥形管中离心8分钟,不超过300×g 。
    3. 用5ml的ACK缓冲液再悬浮脾细胞,并在室温下孵育5分钟。用PBS稀释,并离心8分钟,不超过300×g 。重悬细胞沉淀在FACS缓冲液和计数细胞数。通常,每个脾产生80-100×10 6个脾细胞
    4. 通过使用Dynal小鼠CD4细胞阴性分离试剂盒的阴性选择来纯化CD4 + T细胞,在制造商提供的方案中详细描述。协议产量通常为脾细胞的20-25%( http://tools.invitrogen .com/content/sfs/manuals/dynabeads_untouched_ms_CD4_man.pdf )。
    5. 在CD4 + T细胞分离后,通过离心(在300×g下8分钟)调整细胞浓度,并在FACS缓冲液中稀释至4×10 7个//ml,并进行CD4和CD62L染色
    6. 使用每200μlCD4 + T细胞悬浮液中0.2μg的APC结合的抗CD4抗体和0.3μg的PE缀合的抗CD62L抗体。在4℃下在黑暗中孵育30分钟。
    7. 用FACS缓冲液洗涤细胞,在不超过300×g离心8分钟,并用浓度为3×10 7/ml的FACS缓冲液重悬细胞沉淀。
    8. 使用FACSAria细胞分选仪进行CD4 +高CD62L高级群体的选择和分选。排除使用FSC-H对比FSC-W和SSC-H对SSC-W点图的细胞双联体。 Balb/c小鼠品系的总百分比应在60-70%之间,C57B1/6小鼠品系的总百分比为50-60%。流量建议调整到1-3左右。排序精度可以设置为"yield"。预期分别为每Balb/c和C57Bl/6脾脏约10×10 6次和7×10 6次CD4T初始细胞。

    9. 使用15毫升锥形管收集排序的人口与2.5毫升收集媒体。在使用之前,涡流管使得管壁将被薄层流体覆盖,从而避免细胞死亡 细胞偏转到管。
    10. 保持排序的人口在冰上。

  2. 来自肿瘤细胞的条件培养基
    1. 该步骤可以在Treg分化方案之前的任何时间进行
    2. 将选定的肿瘤细胞在50%汇合的P60皿中用2ml无血清培养基平板。 在37℃下用5%CO 2孵育18小时,然后收集条件培养基。 用0.22μm注射器过滤器过滤,等分成200μl样品并储存在-70℃
  3. 24孔板的抗CD3涂层
    1. 从无菌PBS中的CD3ε抗体(1mg/ml)制备5μg/ml的抗CD3ε溶液并涡旋。 对于24孔板使用每孔150μl
    2. 在37℃在潮湿气氛中孵育至少2小时。
    3. 在使用前,用PBS冲洗孔,并抽吸两次
  4. 在肿瘤细胞条件培养基存在下将天然T细胞转化为Treg
    1. 对于Treg转化不可缺少的刺激是TGFβ1和IL-2。为了评估Gal-1微调Treg转化频率,有必要使用前者的极限浓度。已显示TGFβ1亚型限制在调节Treg分化方面更有效。在补充有1-2ng/mlhTGFβ1,100U/ml mIL-2的无血清RPMI中,将初始T细胞浓度调节至1×10 6/1μg/ml CD28 mAb和抗生素 - 抗真菌剂
    2. 在抗CD3涂覆的24孔板中每孔加入1ml初始T细胞悬浮液
    3. 加入条件培养基(CM)肿瘤细胞。建议确定对CM的剂量依赖性反应。建议稀释度从1:10到1:100。
    4. 在37℃下用5%CO 2孵育4天。更多的日子只会导致细胞死亡增加。
    5. 在CD4,CD25和Foxp3染色后通过流式细胞术检测Treg频率

  5. Treg染色
    1. 在4℃下在黑暗中进行CD4和CD25分子的染色30分钟。 每2×10 6个细胞在100μlFACS缓冲液中使用0.03μg的APC-结合的CD4抗体和0.075μg的AlexaFluor 488-结合的CD25。
    2. 洗涤细胞并在不超过300×g离心8分钟。 固定和透化细胞使用固定/Perm缓冲液100微升30分钟到18小时在黑暗中4°C。
    3. 用1×渗透缓冲液洗涤细胞。 Foxp3染色在100μl1×渗透缓冲液中使用0.225μgPE-缀合的Foxp3抗体在4℃在黑暗中进行1小时。
    4. 用1×渗透缓冲液洗涤细胞,在300×g离心10分钟并重悬在FACS缓冲液中。
    5. 对于流式细胞术分析,必须使用双激光细胞计数器,并且应考虑包含适当补偿样品的5个额外的管。 强烈建议使用FSC-H对比FSC-W和SSC-H对SSC-W点图排除细胞双联体。


  1. 磷酸盐缓冲液(PBS)
    136 mM NaCl 8.2mM Na 2 HPO 4
    1.5mM KH 2 PO 4 4/v/v 2.7mM KCl(pH7.4)
  2. 无菌红血液裂解缓冲液(ACK缓冲液)
    150mM NH 4 Cl/s 10mM KHCO 3
    0.1mM EDTA
    重悬于蒸馏的H 2 O中
  3. FACS缓冲区
    含有0.1%BSA和2mM EDTA的PBS
  4. 分选细胞收集培养基
    补充有20%FBS的RPMI 1640
  5. 重组hTGFβ 1
    溶于磷酸盐缓冲盐水(PBS)(pH 7.4)至工作稀释液30μg/ml分装于-70°C保存
  6. 重组mIL-2


该协议基于在Dalotto-Moreno等人(2013)中发表的原始着作。 这项工作得到了阿根廷国家贸易委员会(ANPCyT; PICT 2007-093到MS和2010-870到GAR和Consejo Nacional de InvestigacionesCientíficasyTécnicas(CONICET; PIP 2010-2012到MS和GAR) ,基金会对GAR的销售作者要特别感谢MaríaRosa Morales的动物技术帮助。


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  2. Dalotto-Moreno,T.,Croci,DO,Cerliani,JP,Martinez-Allo,VC,Dergan-Dylon,S.,Mendez-Huergo,SP,Stupirski,JC,Mazal,D.,Osinaga,E.,Toscano, MA,Sundblad,V.,Rabinovich,GA和Salatino,M。(2013)。 靶向半乳凝素-1可以克服乳腺癌相关的免疫抑制,并预防转移性疾病。 Cancer Res 73(3):1107-1117。
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引用:Dalotto-Moreno, T., Rabinovich, G. A. and Salatino, M. (2014). In vitro Regulatory T cells Differentiation From Naïve T Cells. Bio-protocol 4(6): e1075. DOI: 10.21769/BioProtoc.1075.



Lisa Qin
Hi, Thanks for the detailed protocol. It seems like the Treg differentiation is carried out in serum free medium. Is it OK for T cells? Thanks.
9/17/2015 10:35:35 AM Reply
Marian Salatino
Immunopathology Department, Institute of Biology and Experimental Medicine, CONICET, Argentina

That is correct! This protocol is carried out in serum free medium. Treg differentiation can be carried out like this but it is NOT OK for effector T cells as they will die unless serum is present. You can also differentiate Tregs in medium with serum. We have done it in 2%FCS containing medium and has given us great results. The proteins present in the serum can however retain TGFb and can thus impact negatively on the differentiation efficiency.
Write if you have any other doubts!

9/22/2015 8:30:08 AM