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Ex vivo Co-culture of Lymphoid Tissue Stromal Cells and T Cells
淋巴组织基质细胞和T淋巴细胞的体外共培养   

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Abstract

Stromal cells within lymphoid tissues produce IL-7, which is critical for the survival and function of T cells. This protocol is to be used to isolate primary human lymphoid tissue stromal cells to study their impact on the survival of T cells in an ex vivo co-culture system.

Materials and Reagents

  1. RPMI-1640 medium (Life Technologies, catalog number: 11875-093 )
  2. Fetal bovine serum (FBS) (GEMBIO, catalog number: 900-108 )
  3. Antibiotic-Antimycotic (Life Technologies, catalog number: 15240-062 )
  4. Anti-CD45RA (Dako, catalog number: M0754 )
  5. Anti-activated caspase-3 (Cell Signaling Technology, catalog number: 9665 )
  6. Anti-CD3 (AbD Serotec, catalog number: MCA1477 )
  7. Anti-IL-7 (R&D Systems, catalog number: MAB207 )
  8. Streck's tissue fixative (Streck Laboratories, catalog number: 265138 )
  9. Triton X-100 (Sigma-Aldrich, catalog number: X-100)
  10. CellMicroSieves ( BioDesign Inc. of New York, catalog number: N100S )
  11. Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies, InvitrogenTM, catalog number: A-21206 )
  12. Alexa Fluor 568 Donkey Anti-Mouse IgG (Life Technologies, InvitrogenTM, catalog number: A10037 )
  13. Alexa Fluor 647 Chicken Anti-Rat IgG (Life Technologies, InvitrogenTM, catalog number: A-21472 )
  14. Dimethylsulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  15. Phosphate-Buffered Saline (PBS) (Life Technologies, InvitrogenTM, catalog number: 10010-023 )
  16. Complete RPMI-1640 culture medium (see Recipes)
    Note: The experimental protocols used here for human tissue samples had full IRB approval (Institutional Review Board: Human Subjects Committee, Research Subjects’ Protection Program, University of Minnesota) and informed written consent was obtained from individual patients, or the legal guardians of minors, for the use of tissue in research applications prior to the initiation of surgery.

Equipment

  1. Biological Safety Cabinet
  2. Chamber slides (Thermo Fisher Scientific, catalog number: 154526 )
  3. Centrifuges
  4. Water bath

Procedure

  1. Fresh human palatine tonsil tissues were obtained from routine tonsillectomies and processed within 1–2 h of completion of surgery. Viable tonsil lymphocyte suspensions were prepared by forcing cut tissue pieces through a metal sieve (e.g. CellMicroSieves), and collecting the released single cell suspension in complete RPMI-1640 culture medium.
  2. The lymphocytes were washed twice (centrifuge and resuspend the cells in PBS) and immediately cryopreserved by resuspending cells in freezing medium (FBS with 10% DMSO). The cells were immediately transferred to -20 °C for one hour, followed by -80 °C overnight before permanent storage in liquid nitrogen.
  3. By culturing the stroma left on the metal sieve (e.g. CellMicroSieves), in complete RPMI-1640 culture medium at 37 °C with 5% CO2, adherent proliferating fibroblast-like stromal cells were first visible after 2-5 days in the culture, and confluent monolayers developed after 10-25 days.
  4. For co-culture of lymphocytes and stromal cells, 2 x 105 lymphocytes isolated from human tonsil were cultured in chamber slides without stromal cells, with autologous stromal cells (2 x 104 cells/well), or with autologous stromal cells (2 x 104 cells/well) and IL-7 blocking antibody (50 μg/ml) for 2 to 3 days.
  5. After co-culture, the slides were fixed in 0.5 ml Streck's tissue fixative for 20 min, permeabilized with 1% TritonX-100 for for 5 min.
  6. The slides were washed with 500 μl/well PBS + 3% FBS for 3 times, blocked with 500 μl/well DPBS + 3% FBS + 0.5% Tween-20 for 2 h at room temperature.
  7. After removing the blocking reagent, add 250 μl/well primary antibodies [activated caspase3 (1:100 dilution), CD45RA (1:100 dilution) and CD3 (1:100 dilution)] and incubate at room temperature for 1 h.
  8. After removing the primary antibodies, the slides were washed with 500 μl/well PBS + 3% FBS for 3 times.
  9. Add 250 μl/well fluorescent secondary antibodies and incubate at room temperature for 1 h.
  10. Remove the secondary antibodies and wash the slides with washed with 500 μl/well PBS + 3% FBS for 3 times.
  11. Use fluorescence microscope or confocoal microscope to quantify the number of apoptotic naive T cells.

Recipes

  1. Complete RPMI-1640 culture medium
    Supplemented with 10% heat inactivated fetal calf serum FBS and 1x Antibiotic-Antimycotic

Acknowledgments

This protocol is adapted from Link et al. (2007) and Zeng et al. (2012).

References

  1. Link, A., Vogt, T. K., Favre, S., Britschgi, M. R., Acha-Orbea, H., Hinz, B., Cyster, J. G. and Luther, S. A. (2007). Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells. Nat Immunol 8(11): 1255-1265.
  2. Zeng, M., Southern, P. J., Reilly, C. S., Beilman, G. J., Chipman, J. G., Schacker, T. W. and Haase, A. T. (2012). Lymphoid tissue damage in HIV-1 infection depletes naive T cells and limits T cell reconstitution after antiretroviral therapy. PLoS Pathog 8(1): e1002437.

简介

淋巴组织内的基质细胞产生IL-7,其对于T细胞的存活和功能至关重要。 该方案用于分离原代人淋巴样组织基质细胞,以研究其对离体共培养系统中T细胞存活的影响。

材料和试剂

  1. RPMI-1640培养基(Life Technologies,目录号:11875-093)
  2. 胎牛血清(FBS)(GEMBIO,目录号:900-108)
  3. 抗生素 - 抗真菌剂(Life Technologies,目录号:15240-062)
  4. 抗CD45RA(Dako,目录号:M0754)
  5. 抗激活的胱天蛋白酶-3(Cell Signaling Technology,目录号:9665)
  6. 抗CD3(AbD Serotec,目录号:MCA1477)
  7. 抗IL-7(R& D Systems,目录号:MAB207)
  8. Streck组织固定剂(Streck Laboratories,目录号:265138)
  9. TritonX-100(Sigma-Aldrich,目录号:X-100)
  10. Cell MicroSieves(New York的BioDesign Inc.,目录号:N100S)
  11. Alexa Fluor 488驴抗兔IgG(Life Technologies,Invitrogen TM,目录号:A-21206)
  12. Alexa Fluor 568驴抗小鼠IgG(Life Technologies,Invitrogen TM ,目录号:A10037)
  13. Alexa Fluor 647鸡抗大鼠IgG(Life Technologies,Invitrogen TM ,目录号:A-21472)
  14. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  15. 磷酸盐缓冲盐水(PBS)(Life Technologies,Invitrogen TM ,目录号:10010-023)
  16. 完成RPMI-1640培养基(见配方)
    注意:这里用于人组织样品的实验方案具有完全的IRB批准(Institutional Review Board:Human Subjects Committee,Research Subjects'Protection Program,University of Minnesota),并且从个体患者获得书面同意,或者合法 未成年人监护人,在开始手术前在研究应用中使用组织。

设备

  1. 生物安全柜
  2. 室玻片(Thermo Fisher Scientific,目录号:154526)
  3. 离心机
  4. 水浴

程序

  1. 新鲜人腭扁桃体组织从常规扁桃体获得,并在手术完成的1-2小时内加工。通过迫使切割的组织片通过金属筛(例如CellMicroSieves)并将释放的单细胞悬浮液收集在完全RPMI-1640培养基中来制备活的扁桃体淋巴细胞悬浮液。
  2. 将淋巴细胞洗涤两次(离心并将细胞重悬于PBS中),并通过将细胞重悬于冷冻培养基(具有10%DMSO的FBS)中立即冷冻保存。将细胞立即转移至-20℃1小时,然后在-80℃下过夜,然后永久保存在液氮中。
  3. 通过在完全RPMI-1640培养基中在37℃下用5%CO 2 2培养留在金属筛(例如CellMicroSieves)上的基质,贴壁增殖成纤维细胞样基质细胞在培养物中2-5天后首先可见,并且在10-25天后形成汇合的单层
  4. 对于淋巴细胞和基质细胞的共培养,将从人扁桃体分离的2×10 5个淋巴细胞在没有基质细胞的室玻片中与自体基质细胞(2×10 4个/(2×10 4个细胞/孔)和IL-7阻断抗体(50μg/ml)处理2〜3天。
  5. 共培养后,将载玻片在0.5ml Streck组织固定液中固定20分钟,用1%TritonX-100透化5分钟。
  6. 载玻片用500μl/孔PBS + 3%FBS洗涤3次,用500μl/孔DPBS + 3%FBS + 0.5%Tween-20在室温下封闭2小时。
  7. 除去封闭试剂后,加入250μl/孔一抗[活化的胱天蛋白酶3(1:100稀释),CD45RA(1:100稀释)和CD3(1:100稀释)],并在室温下孵育1小时。 />
  8. 除去第一抗体后,载玻片用500μl/孔PBS + 3%FBS洗涤3次
  9. 加入250μl/孔荧光第二抗体,并在室温下孵育1小时
  10. 取出二抗,洗涤载玻片,用500μl/孔PBS + 3%FBS洗涤3次
  11. 使用荧光显微镜或共焦显微镜量化凋亡幼稚T细胞的数量

食谱

  1. 完成RPMI-1640培养基
    补充10%热灭活的胎牛血清FBS和1×抗生素 - 抗真菌剂

致谢

该协议改编自Link等人。 (2007)和Zeng 等人(2012)。

参考文献

  1. Link,A.,Vogt,T.K.,Favre,S.,Britschgi,M.R.,Acha-Orbea,H.,Hinz,B.,Cyster,J.G.and Luther,S.A。(2007)。 淋巴结中的成纤维网状细胞调节幼稚T细胞的内环境稳定。 Nat Immunol 8(11):1255-1265
  2. Zeng,M.,Southern,P.J.,Reilly,C.S.,Beilman,G.J.,Chipman,J.G.,Schacker,T.W.and Haase,A.T。 HIV-1感染中的淋巴组织损伤会消耗幼稚的T细胞,并限制抗逆转录病毒治疗后的T细胞重建。 PLoS Pathog 8(1):e1002437。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Zeng, M. and Haase, A. T. (2012). Ex vivo Co-culture of Lymphoid Tissue Stromal Cells and T Cells. Bio-protocol 2(17): e255. DOI: 10.21769/BioProtoc.255.
  2. Zeng, M., Southern, P. J., Reilly, C. S., Beilman, G. J., Chipman, J. G., Schacker, T. W. and Haase, A. T. (2012). Lymphoid tissue damage in HIV-1 infection depletes naive T cells and limits T cell reconstitution after antiretroviral therapy. PLoS Pathog 8(1): e1002437.
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