In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay
使用体外脉冲树突状细胞体外致敏T细胞: 腘窝淋巴结实验   

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Stem Cell Reports
May 2017



One-way mixed lymphocyte reaction (MLR) is a classic tool to measure how T cells react to external stimuli. However, MLR is an in vitro reaction system, which shows different response intensity compared with in vivo trails sometimes due to the lack of cytokines, tissue matrix and other immune response associated factors. The following popliteal lymph node assay (PLNA) protocol is designed to test the T cells antigen-specific reaction in vivo by using ovalbumin (OVA) specific reacted transgenic mouse OT-1 and OT-2.

Keywords: Mixed lymphocyte reaction (MLR) (混合淋巴细胞反应(MLR)), Popliteal lymph node assay (PLNA) (腘窝淋巴结实验(PLNA)), Antigen presenting cells (APC) (抗原呈递细胞(APC)), Dendritic cells (DC) (树突状细胞(DC)), Flow cytometry (流式细胞仪), Ovalbumin antigen (卵清蛋白抗原), OT-1 (OT-1), OT-2 (OT-2)


In transplantation, autoimmune and infection studies, one-way mixed lymphocyte reaction (MLR) is a helpful parameter to assess whether T-cell proliferation is increased or inhibited in response to antigen present cells (APC) or other external stimuli. This is a classic functional test, which demonstrates how the T-cell is affected by the test reagents, such as immune-suppress drug (Hou et al., 2015; Zhang et al., 2015), negative cell or exosome vaccines (Ma et al., 2016; Cai et al., 2017).

However, this system has some limits due to the fact that it is in vitro immune reaction system. In vivo immune reaction is affected by bio-microenvironment, such as cytokines, chemokines, hormones secretion, tissue matrix and other unknown bio-factors.

Herein, the popliteal lymph node assay (PLNA) was designed as an in vivo tool to test the increased or decreased immune response by using an ovalbumin (OVA) antigen-specific reaction system (Bhatt et al., 2014; Cai et al., 2017).

Materials and Reagents

  1. Gloves, mask and lab coat
  2. Pipette tips
    10 μl (FUKAEKASEI and WATSON, catalog number: 110-207C )
    200 μl (FUKAEKASEI and WATSON, catalog number: 110-705C )
    1 ml (FUKAEKASEI and WATSON, catalog number: 110-706C )
  3. 27 G needle (Terumo Medical, catalog number: NN-2732R )
  4. 24-well tissue culture plate (Greiner Bio One International, CELLSTAR®, catalog number: 662160 )
  5. Nylon fiber column T (Wako Pure Chemical Industries, catalog number: 147-06721 )
  6. 21 G needle (Terumo Medical, catalog number: NN-2138R )
  7. Nylon mesh, 70 μm (AS ONE, catalog number: PA-70μ )
  8. Culture dish
    3.5 ml (Greiner Bio One International, CELLSTAR®, catalog number: 627170 )
    10 ml (Greiner Bio One International, CELLSTAR®, catalog number: 664160 )
  9. BD Falcon® disposable transfer pipets, 3 ml (Corning, Falcon®, catalog number: 357524 )
  10. 10 ml serological pipette (Greiner Bio One International, CELLSTAR®, catalog number: 607180 )
  11. Falcon® 15 ml conical centrifuge tubes (Corning, Falcon®, catalog number: 352097 )
  12. 1.5 ml round-bottom micro tube (FUKAEKASEI and WATSON, catalog number: 131-715C )
  13. Aluminum foil (Mitsubishi)
  14. C57BL6/Nslc (B6) mice
  15. 70% ethanol (Wako Pure Chemical Industries, catalog number: 324-00037 )
  16. Crashed ice and carrier
  17. Recombinant Murine GM-CSF (PeproTech, catalog number: 315-03 )
  18. Recombinant Murine IL-4 (PeproTech, catalog number: 214-14 )
  19. Recombinant Murine IFN-γ (PeproTech, catalog number: 315-05 )
  20. EndoGrade® Endotoxin-free ovalbumin (Hyglos, catalog number: 321000 )
  21. Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230162 )
  22. Phosphate-buffered saline (PBS; 10x), pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 70013073 )
  23. CellTraceTM Violet Cell Proliferation Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34557 )
  24. CellTraceTM CFSE Cell Proliferation Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: C34554 )
  25. APC anti-mouse CD4 Antibody (BioLegend, catalog number: 100516 )
  26. APC Rat IgG2a, κ Isotype Ctrl Antibody (isotype) (BioLegend, catalog number: 400511 )
  27. PE anti-mouse CD8a Antibody (BioLegend, catalog number: 100708 )
  28. PE Rat IgG2a, κ Isotype Ctrl Antibody (isotype) (BioLegend, catalog number: 400507 )
  29. RPMI-1640 (Wako Pure Chemical Industries, catalog number: 189-02025 )
  30. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140079 )
  31. 100x penicillin-streptomycin-glutamine (Thermo Fisher Scientific, catalog number: 10378016 )
  32. 100x MEM non-essential amino acids solution (Thermo Fisher Scientific, GibcoTM, catalog number: 11140 )
  33. 100 mM sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  34. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  35. Complete medium (see Recipes)


  1. Sanyo Bio clean bench (Panasonic Healthcare, catalog number: MCV-B131F )
  2. CO2 incubator (Panasonic Healthcare, catalog number: MCO-5ACUV-PJ )
  3. Autoclave (TOMY SEIKO, model: ES-315 )
  4. Tweezers (Weller, catalog number: 4SA )
  5. Centrifuge (Beckman Coulter, catalog number: Allegra X-12 )
  6. Water bath (Tokyo Rikakikai, EYELA, model: NTT-2000 )
  7. Dissecting instrument start set (NATSUME SEISAKUSHO, Nazme, catalog number: E-16-F )
  8. Flow cytometer (Beckman Coulter, model: Gallios )


  1. FlowJo V.10.0.8. (FlowJo, LLC)


  1. Dendritic cells (DCs) culture and antigen loading (Figure 1)
    1. C57BL6/Nslc (B6) mice are kept within a CO2 chamber for 5 min and are confirmed dead. Both ends of the femoral and tibial bone cortex are drilled with a 27 G needle. 5 ml PBS is injected through the hole of one end, and bone marrow cells (BMCs) can be collected from the hole of the other end. About 5-8 x 107 BMCs can be collected from one mouse. BMCs are cultured in a 24-well TC plate as 1 x 106 cells per well in 1 ml complete medium (see Recipes) in the presence of GM-CSF (10 ng/ml), IL-4 (10 ng/ml) in 5% CO2 in air at 37 °C.
    2. On Day 2, the culture soup is removed and new complete medium with GM-CSF (10 ng/ml), IL-4 (10 ng/ml) is added to the plate (0.5 ml per well). The culture soup is centrifuged (300 x g, 5 min, 22 °C) and the supernatant is added back to the plate (0.5 ml per well).
    3. On Day 5, the cells are harvested and washed 2 times with PBS. The split ratio is about 60-80%. Cells are cultured into a new 24-well TC plate as 1 x 106 per well in 1 ml complete medium in the presence of GM-CSF (10 ng/ml), IL-4 (10 ng/ml) and IFN-γ (10 ng/ml). The final volume is 1 ml per well.
    4. On Day 6, the cells are pulsed with OVA (400 μg/ml) for 24 h. In detail, OVA is diluted in PBS at 100 μg/μl first and then added into DCs at 4 μl per well. The final volume is 1.004 ml per well. At least 2 wells of cells are untreated with OVA as negative control for the reaction.
    5. Cells are harvested on Day 7. Because the cells of interest are loosely adherent, we 1) tap the plate gently; 2) harvest the fluid; 3) flush the empty well with 1 ml PBS and collect the fluid. The harvested cells are washed with PBS three times to make sure that the redundant OVA is removed thoroughly. Cells are suspended in PBS at 1.5-4.5 x 105 per 30 μl for use (Figure 1).

      Figure 1. OVA pulsed DCs preparation. Bone marrow cells are isolated from B6 mice and cultured in completed medium with GM-CSF and IL-4 for 7 days. Half/half medium is changed on Day 2. BM-DCs are stimulated with IFN-γ on Day 5 for 48 h and are pulsed with OVA on Day 6 for 24 h. Cells are harvested on Day 7 and washed thoroughly before using.

  2. T cells preparation
    1. Spleens are isolated from OT-1 and OT-2 Tg mice. Spleen cells are hemolyzed in 4.5 ml distilled water for 6 sec followed by 0.5 ml 10x PBS. After this, 5 ml 1x PBS is added to dilute the suspension.
    2. Spleen T cells are separated by using Nylon Column T. Nylon Column T is washed by 10 ml pre-warmed RPMI-1640 slowly. Air is removed by squeezing the nylon fiber. Hemolyzed spleen cells (< 1 x 108) are suspended in 1 ml of warm RPMI and loaded onto the column. The T-shape stopcock, which connected the column and 21 G needle, is switched to let the suspension infiltrate into the nylon fiber followed by 1 ml additional RPMI-1640 loading onto the column. Then, the column is sealed and incubated at 37 °C and 5% CO2 for 60 min. After that, T cells are then eluted with 5 ml warm RPMI.
    3. T cells are labeled with CellTrace Violet or CFSE Cell Proliferation Kit (2.5 μM, 37 °C, 8 min) according to product manual. Violet or CFSE labeled T cells are suspended in PBS at 3 x 106/500 μl for use. Details are shown in Figure 2.

      Figure 2. T cells isolation and labeling. Spleen cells are hemolyzed with distilled water and 10x PBS. T cells are separated by nylon-wool column and then labeled with Cell-Trace CFSE or Violet.

  3. Popliteal lymph node assay (PLNA)
    1. Violet or CFSE labeled OT-1 or OT-2 T cells are adoptively transferred intravenously to naïve B6 mice (recipients) at 3 x 106/500 μl/mice at Day -1. Then OVA pulsed BMDCs are injected subcutaneously into the recipients’ footpads at 4.5 x 105/0.03 ml at Day 0. The popliteal lymph nodes (PLNs) are harvested at Day 3.
    2. Lymph nodes (LNs) capsules are pierced by 27 G needle. Cells isolated from LN are filtered with 70 μm mesh and stained with CD8-PE (violet labeled T cells group) or CD4-APC (CFSE labeled group) for flow cytometry assessment. Details are shown in Figure 3.

      Figure 3. PLNA procedure. Step 1. Cell-Trace labeled T cells are transferred into wide type B6 at Day -1. OVA-loaded DCs are injected subcutaneously into their footpads at Day 0. The popliteal lymph nodes (PLNs) are harvested at Day 3. Step 2. Cells harvested from PLNs are stained with antibody and detected by flow cytometry.

Data analysis

  1. Cells isolated from PLN are detected by Gallios flow cytometer and analyzed with FlowJo V.10.0.8.
  2. Proliferation of T cells is determined by Violet or CFSE dilution gated on CD8+ (OT-I) or CD4+ (OT-II) population. Grey lines indicate PBS injection into the footpad (i.fp), used as a negative control (Figure 4).

    Figure 4. PLNA data. CD8+Violet+ (OT-1) or CD4+CFSE+ (OT-II) double positive population is gated for a histogram view. T cells transferred + PBS i.fp, negative control group (grey) indicates the original non-proliferation population. The proliferated population is gated and shown as a percentage of total T cells.


  1. Complete medium (500 ml)
    435 ml RPMI-1640
    50 ml GibcoTM fetal bovine
    5 ml 100x penicillin-streptomycin-glutamine
    5 ml 100x GibcoTM MEM non-essential amino acids
    5 ml 100 mM GibcoTM sodium pyruvate
    2 μl 2-mercaptoethanol


This study was supported by research grants from the National Center for Child Health and Development (26-6, 26-27 and 27-21), Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid 15F15756 and 15K10043).


  1. Bhatt, S., Qin, J., Bennett, C., Qian, S., Fung, J. J., Hamilton, T. A. and Lu, L. (2014). All-trans retinoic acid induces arginase-1 and inducible nitric oxide synthase-producing dendritic cells with T cell inhibitory function. J Immunol 192(11): 5098-5108.
  2. Cai, S., Hou, J., Fujino, M., Zhang, Q., Ichimaru, N., Takahara, S., Araki, R., Lu, L., Chen, J. M., Zhuang, J., Zhu, P. and Li, X. K. (2017). iPSC-derived regulatory dendritic cells inhibit allograft rejection by generating alloantigen-specific regulatory T cells. Stem Cell Reports 8(5): 1174-1189.
  3. Hou, J., Zhang, Q., Fujino, M., Cai, S., Ito, H., Takahashi, K., Abe, F., Nakajima, M., Tanaka, T., Xu, J., Zou, H., Ding, Q. and Li, X. K. (2015). 5-Aminolevulinic acid with ferrous iron induces permanent cardiac allograft acceptance in mice via induction of regulatory cells. J Heart Lung Transplant 34(2): 254-263.
  4. Ma, B., Yang, J. Y., Song, W. J., Ding, R., Zhang, Z. C., Ji, H. C., Zhang, X., Wang, J. L., Yang, X. S., Tao, K. S., Dou, K. F. and Li, X. (2016). Combining exosomes derived from immature DCs with donor antigen-specific Treg cells induces tolerance in a rat liver allograft model. Sci Rep 6: 32971.
  5. Zhang, Q., Ichimaru, N., Higuchi, S., Cai, S., Hou, J., Fujino, M., Nonomura, N., Kobayashi, M., Ando, H., Uno, A., Sakurai, K., Mochizuki, S., Adachi, Y., Ohno, N., Zou, H., Xu, J., Li, X. K. and Takahara, S. (2015). Permanent acceptance of mouse cardiac allografts with CD40 siRNA to induce regulatory myeloid cells by use of a novel polysaccharide siRNA delivery system. Gene Ther 22(3): 217-226.


单向混合淋巴细胞反应(MLR)是测量T细胞如何与外部刺激反应的经典工具。 然而,MLR是体外反应系统,其与体内踪迹相比显示出不同的反应强度,有时由于缺乏细胞因子,组织基质和其他免疫应答相关因素。 设计了以下po淋巴结测定(PLNA)方案,通过使用卵白蛋白(OVA)特异性反应的转基因小鼠OT-1和OT-2,体内测试T细胞抗原特异性反应。
【背景】在移植中,自身免疫和感染研究中,单向混合淋巴细胞反应(MLR)是评估T细胞增殖在抗原存在细胞(APC)或其他外部刺激反应中是否增加或抑制的有用参数。 这是一个典型的功能测试,它展示了T细胞如何受到免疫抑制药物(Hou et al。,2015; Zhang et al。,2015),阴性细胞或外来体疫苗(Ma)的测试试剂的影响 et al。,2016; Cai et al。,2017)。
   然而,由于体外免疫反应系统,该系统具有一定的限制。 体内免疫反应受生物微环境的影响,如细胞因子,趋化因子,激素分泌,组织基质等未知生物因子。
   在这里,po淋巴结测定(PLNA)被设计为体内工具,通过使用卵白蛋白(OVA)抗原特异性反应系统来测试增加或减少的免疫应答(Bhatt等,2014; Cai et al。,2017)。

关键字:混合淋巴细胞反应(MLR), 腘窝淋巴结实验(PLNA), 抗原呈递细胞(APC), 树突状细胞(DC), 流式细胞仪, 卵清蛋白抗原, OT-1, OT-2


  1. 手套,面具和实验室外套
  2. 移液器提示
  3. 27 G针(Terumo Medical,目录号:NN-2732R)
  4. 24孔组织培养板(Greiner Bio One International,CELLSTAR ,目录号:662160)
  5. 尼龙纤维柱T(和光纯药,目录号:147-06721)
  6. 21 G针(Terumo Medical,目录号:NN-2138R)
  7. 尼龙网,70μm(AS ONE,目录号:PA-70μ)
  8. 文化盘
    3.5 ml(Greiner Bio One International,CELLSTAR ®,目录号:627170)
    10 ml(Greiner Bio One International,CELLSTAR ®,目录号:664160)
  9. BD Falcon ®一次性转移移液管,3 ml(Corning,Falcon ®,目录号:357524)
  10. 10 ml血清移液管(Greiner Bio One International,CELLSTAR ®,目录号:607180)
  11. Falcon ®将15ml锥形离心管(Corning,Falcon ®,目录号:352097)
  12. 1.5毫升圆底微管(FUKAEKASEI和WATSON,目录号:131-715C)
  13. 铝箔(三菱)
  14. C57BL6 / Nslc(B6)小鼠
  15. 70%乙醇(Wako Pure Chemical Industries,目录号:324-00037)
  16. 坠冰和载体
  17. 重组鼠GM-CSF(PeproTech,目录号:315-03)
  18. 重组鼠IL-4(PeproTech,目录号:214-14)
  19. 重组小鼠IFN-γ(PeproTech,目录号:315-05)
  20. EndoGrade ®无内毒素卵白蛋白(Hyglos,目录号:321000)
  21. 蒸馏水(Thermo Fisher Scientific,Gibco TM ,目录号:15230162)
  22. 磷酸缓冲盐水(PBS; 10x),pH7.2(Thermo Fisher Scientific,Gibco TM,目录号:70013073)
  23. CellTrace TM紫细胞增殖试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:C34557)
  24. CellTrace TM CFSE细胞增殖试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:C34554)
  25. APC抗小鼠CD4抗体(BioLegend,目录号:100516)
  26. APC大鼠IgG2a,κ同种型Ctrl抗体(同种型)(BioLegend,目录号:400511)
  27. PE抗小鼠CD8a抗体(BioLegend,目录号:100708)
  28. PE大鼠IgG2a,κ同种型Ctrl抗体(同种型)(BioLegend,目录号:400507)
  29. RPMI-1640(和光纯药,目录号:189-02025)
  30. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:26140079)
  31. 100x青霉素 - 链霉素 - 谷氨酰胺(Thermo Fisher Scientific,目录号:10378016)
  32. 100x MEM非必需氨基酸溶液(Thermo Fisher Scientific,Gibco TM,目录号:11140)
  33. 100mM丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  34. 2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  35. 完成培养基(见食谱)


  1. 三洋生物洁净台(松下医疗保健,目录号:MCV-B131F)
  2. CO 2培养箱(Panasonic Healthcare,目录号:MCO-5ACUV-PJ)
  3. 高压灭菌器(TOMY SEIKO,型号:ES-315)
  4. 镊子(Weller,目录号:4SA)
  5. 离心机(Beckman Coulter,目录号:Allegra X-12)
  6. 水浴(东京Rikakikai,EYELA,型号:NTT-2000)
  7. 解剖仪开始设定(NATSUME SEISAKUSHO,Nazme,目录号:E-16-F)
  8. 流式细胞仪(Beckman Coulter,型号:Gallios)


  1. FlowJo V.10.0.8。 (FlowJo,LLC)


  1. 树突状细胞(DCs)培养和抗原加载(图1)
    1. 将C57BL6 / Nslc(B6)小鼠保持在CO 2室中5分钟,并确认死亡。股骨和胫骨骨皮质两端均用27 G针刺。通过一端的孔注入5ml PBS,从另一端的孔收集骨髓细胞(BMC)。约5-8 x 10 7 可以从一只小鼠收集BMC。在GM-CSF(10ng / ml),IL-1β的存在下,在1ml完全培养基(见Recipes)中,将BMC在24孔TC板中培养1×10 6个细胞/孔, 4(10ng / ml)在5%CO 2 /空气中在37℃。
    2. 在第2天,除去培养汤,并向板中加入新的GM-CSF(10ng / ml),IL-4(10ng / ml)的完全培养基(0.5ml /孔)。将培养汤离心(300×g,5分钟,22℃),将上清液加回板中(每孔0.5ml)。
    3. 在第5天,收获细胞并用PBS洗涤2次。分流比约为60-80%。在GM-CSF(10ng / ml),IL-4(10ng / ml)存在下,将细胞在1ml完全培养基中,每孔1×10 6个/孔/ ml)和IFN-γ(10ng / ml)。最终体积为1ml /孔。
    4. 在第6天,细胞用OVA(400μg/ ml)脉冲24小时。详细地,首先用100μg/μl的PBS稀释OVA,然后以4μl/孔加入到DC中。最终体积为1.004ml /孔。至少2口细胞未用OVA作为阴性对照进行反应。
    5. 细胞在第7天被收获。因为感兴趣的细胞松散粘附,我们1)轻轻地敲击板; 2)收获液体; 3)用1ml PBS冲洗空孔并收集流体。将收获的细胞用PBS洗涤三次,以确保彻底去除多余的OVA。将细胞以每30μl1.5-4.5×10 5个/ ml悬浮于PBS中(图1)。

      图1. OVA脉冲DCs制备从B6小鼠中分离骨髓细胞,并在含GM-CSF和IL-4的完全培养基中培养7天。在第2天改变半/半培养基。在第5天用IFN-γ刺激BM-DC 48小时,并在第6天用OVA脉冲24小时。细胞在第7天收获,并在使用前彻底清洗。

  2. T细胞制备
    1. 脾从OT-1和OT-2 Tg小鼠分离。脾细胞在4.5ml蒸馏水中溶血6秒,然后加入0.5ml 10x PBS。之后,加入5ml 1x PBS稀释悬浮液。
    2. 使用尼龙柱T分离脾T细胞。尼龙柱T缓慢用10ml预热的RPMI-1640洗涤。通过挤压尼龙纤维除去空气。将溶血的脾细胞(<1×10 8)悬浮在1ml温的RPMI中并加载到柱上。将连接色谱柱和21 G针的T型旋塞旋转,使悬浮液浸入尼龙纤维中,然后将1ml额外的RPMI-1640装载到柱上。然后,将柱密封并在37℃和5%CO 2下孵育60分钟。之后,用5ml温热RPMI洗脱T细胞
    3. 根据产品手册,T细胞用CellTrace Violet或CFSE细胞增殖试剂盒(2.5μM,37℃,8分钟)标记。将紫色或CFSE标记的T细胞以3×10 6 /500μl/500μl悬浮于PBS中用于使用。详细信息如图2所示

      图2. T细胞分离和标记。脾细胞用蒸馏水和10x PBS溶血。 T细胞由尼龙羊毛柱分离,然后用Cell-Trace CFSE或紫色标记。

  3. al淋巴结检测(PLNA)
    1. 紫外线或CFSE标记的OT-1或OT-2 T细胞在第-1天以3×10 6 /500μl/小时静脉内转移到幼稚的B6小鼠(受体)。然后将OVA脉冲BMDCs在第0天以4.5×10 5 /0.03ml皮下注射到受体的脚垫中。在第3天收获po淋巴结(PLN)。
    2. 淋巴结(LN)胶囊被27 G针刺穿。用70μm筛网过滤从LN分离的细胞,用CD8-PE(紫色标记的T细胞组)或CD4-APC(CFSE标记的组)染色用于流式细胞术评估。详细信息如图3所示



  1. 通过Gallios流式细胞仪检测从PLN分离的细胞,并用FlowJo V.10.0.8进行分析
  2. 通过在CD8 +(OT-1)或CD4 +(OT-II)群体上门控的紫罗兰或CFSE稀释来测定T细胞的增殖。灰线表示PBS注入足垫(i.fp),用作阴性对照(图4)

    图4. PLNA数据。 CD8 + Violet + (OT-1)或CD4 + CFSE + (OT-II)双正数人口选择直方图视图。 T细胞转移+ PBS i.fp,阴性对照组(灰色)表示原始的不扩散群体。增殖群体门控并显示为总T细胞的百分比。


  1. 完全介质(500 ml)
    435 ml RPMI-1640
    50ml Gibco TM 胎牛
    5 ml 100x青霉素 - 链霉素 - 谷氨酰胺
    5 ml 100x Gibco MEM非必需氨基酸
    5毫升100毫摩尔Gibco 丙酮酸钠




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引用:Cai, S., Fujino, M., Lu, L. and Li, X. (2017). In vivo Priming of T Cells with in vitro Pulsed Dendritic Cells: Popliteal Lymph Node Assay. Bio-protocol 7(17): e2531. DOI: 10.21769/BioProtoc.2531.