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Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions
流动状态下T淋巴细胞对肿瘤细胞的粘着强度分析   

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Abstract

This method allows evaluating the relative adhesion strength between T lymphocytes and specific adherent target cells using a shear force in flow chambers. It is based on the measure of the resistance of conjugates formed between T cells and adherent tumor cells to shear stress in a microfluidic system. For this purpose, T cells, stained with a CellTracker probe, are added into flow channels containing a monolayer of adherent target cells and their progressive detachment under a constant shear stress is then recorded using a fluorescent microscope.

Material and Reagents

  1. Adherent tumor cells [such as non-small cell lung carcinoma (NSCLC) cell lines]
  2. Specific T-cell clones (generated either from autologous tumor-infiltrating T lymphocytes (TIL) or peripheral blood lymphocytes (PBL))
  3. RPMI 1640 (Life Technologies, Gibco®, catalog number: 61870044 )
  4. DMEM-F12 (Life Technologies, Gibco®, catalog number: 31331093 )
  5. UltroserG (Pall, catalog number: 15950-017 )
  6. Fetal Bovin Serum (Life Technologies, Gibco®, catalog number: 10270-106 )
  7. Human serum AB (Institut de Biotechnologies Jacques Boy)
  8. Penicillin and streptomycin (Life Technologies, Gibco®, catalog number: 15140122 )
  9. Sodium Pyruvate (Life Technologies, Gibco®, catalog number 11360029 )
  10. IL-2
  11. 10x PBS (Life Technologies, Gibco®, catalog number: 70011-036 )
  12. CellTracker probe (CellTrackerTM Green CMFDA) (Life Technologies, Invitrogen™, catalog number: C2925 )
  13. Complete DMEM: tumor cell culture medium (LC medium) (see Recipes)
  14. RPMI-based T-cell complete medium (see Recipes)

Equipment

  1. Microscope Zeiss LSM-510 (ZEISS) with a heated incubation chamber and CO2 supply
  2. Micro-Slides VI, ibiTreat (ibidi GmbH, catalog number: 80606 ), two silicon tubes (1.6 mm of inner diameter) with a plastic clip, two Elbow Luer connectors (ibidi GmbH, catalog number: 80646 )
  3. Syringe pump (high flow rate > 50 ml/min)
  4. 60 ml syringe (Becton, Dickinson and Company, catalog number: 300866 )
  5. Humidified incubator at 37 °C with 5% CO2
  6. A recipient for waste flow buffer (Erlenmeyer)
  7. Centrifuge (Beckman Coulter, model: GS-6R )

Procedure

  1. Adherent tumor cell preparation
    1. Seed adherent tumor cells into IBIDI channels by adding 60 μl of tumor cell suspension in LC medium. Micro-slides VI, ibiTreat characteristics are the following:
      Number of channels: 6
      Minimal volume per channel: 30 μl
      Channel length: 17 mm
      Channel width: 3.8 mm
      Channel height: 0.4 mm
      Growth area: 0.6 cm2 per channel
      Tumor cell concentration may vary according to the cell type (for instance: 1.6 x 106 cells/ml for NSCLC cell lines described in Reference 1). Cells should be at 90-95% of confluence the day of experiment.
    2. Incubate the IBIDI slide in a humidified incubator at 37 °C for at least 2 h for cell attachment.
    3. Fill gently the reservoirs with another 60 μl of LC medium. Avoid pipetting directly into the channels not to detach the cells.
    4. Incubate overnight at 37 °C, 5% CO2.
      Note: In case of tumor cell treatment (example siRNA transfection), cells should be plated two days before using the same experimental conditions. Medium may need to be changed every 24 h. Be sure that the cells are all alive and just reaching 90-95% confluence the day of experiment.

  2. T-cell preparation
    1. Wash T cells with PBS 1x by centrifugation at 350 x g for 5 min.
    2. Stain cells with CellTracker Green (CMFDA) according to the manufacturer’s protocol. Briefly, resuspend cells at 2 x 106/ml in PBS and add one volume of CellTracker Green (CMFDA 2x) diluted in PBS (final concentration 1 μM). Incubate for 15 min at 37 °C.
    3. Wash T cells twice with RPMI-based complete medium by centrifugation at 350 x g, 5 min.
    4. Resuspend T cells in T-cell medium, at final concentration 2 x 106 cells/ml, in 24 flat bottom well plates.
    5. Incubate T cells overnight in humidified incubator at 37 °C with 5% CO2.

  3. T-cell adhesion strength under flow conditions
    1. The following day, warm the thermostatic chamber of the microscope at 37 °C and 5% CO2 (Figure 1A).
    2. Equilibrate RPMI-based complete medium (500 ml) inside the incubator at 37 °C and 5% CO2.
    3. Prepare syringe pump (Figure 1B).
    4. Connect the tube carrying a plastic clip (position closed) to the syringe and fill the syringe with prewarmed medium. Put the Elbow Luer connector and prime the tube (Figure 1A).
    5. Put the IBIDI slide (Figure 1C) under the microscope objective (20x) (Figure 1D). Be sure that the reservoirs are completely full. If not, add some medium.
    6. Connect the tube to one extremity of the IBIDI channel making sure there are no air bubbles remaining inside. This step is critical, because bubbles increase the risk of tumor cell detachment, influence the flow rate and can even stop the flow.
                  


      Figure 1. Flow system. A. Whole flow system; B. Pump system; C. Details of Micro-Slides IBIDI connections; D. During acquisition, the slide is fixed under the microscope and connected to the pump system.

    7. Use the second tube to connect the opposite extremity of the channel with a bottle collecting wastes (Figure 2).


      Red: adherent tumor cell layer
      Green: T cells
      Blue: LC medium
      White arrow: direction of the flow
      Figure 2. T-cell adhesion under flow conditions. Stained T lymphocytes were incubated for 15 min on a monolayer of autologous tumor cells previously seeded into IBIDI channels. The IBIDI slide is then connected by silicon tubes, in one side to a pump (with a syringe filled with prewarmed medium) and in the other side to the waste recipient.

    8. Release the clip on the tube connecting the syringe to the IBIDI slide (Figure 1A).
      Note: Test the system to validate the maximal flow rate that doesn’t detach tumor cells. This rate will be applied to determine the T-cell adhesion strength on tumor cells.
    9. Wash T cells with RPMI by centrifugation at 350 x g for 5 min.
    10. Resuspend T cells in RPMI medium at final concentration 2 x 106 cells/ml.
    11. Replace the medium filling the channels with 50 μl of T cells suspension. Be careful to not detach tumor cells or introduce bubbles inside channels.
    12. Incubate 15 min at 37 °C.
    13. After incubation, prepare the flow system using the same conditions described for the test assay.
    14. Add 50 ml pre-warmed medium inside the syringe.
    15. Start the acquisition just before the flow (Figure 1D).
    16. Acquire images every 2 s for 60 s. It is expected that T cells adhere more firmly to tumor cells that express adherence molecules (such as ligands for integrins expressed by T cells) than tumor cells that do not express these molecules. (Figure 3)


      Figure 3. Representative images acquired at different time lapses during T cell adhesion protocol. A. 0 sec; B. 250 sec; C. 640 sec at the flow rate of 100 ml/h.

Recipes

  1. Complete DMEM: tumor cell culture medium (LC)
    DMEM-F12 supplemented with:
    10% decomplemented Fetal Bovine Serum
    1% UltroserG
    1% Penicillin and streptomycin
    1% Sodium pyruvate
  2. RPMI-based T-cell complete medium
    RPMI 1640 complemented with 10% Human serum AB
    1% Penicillin and streptomycin
    1% Sodium pyruvate
    IL-2 (100 U/ml)

Acknowledgments

We thank Sophie Salomé-Desmoulez for her help with confocal microscopy. This work was supported by grants from the INSERM, the Association pour la Recherche sur le Cancer (ARC), the Institut National du Cancer (INCa), the Ligue contre le Cancer and the Cancéropôle Ile de France (IDF). MB is a recipient of a fellowship from the Cancéropôle IDF.

References

  1. Bernard, G., Raimondi, V., Alberti, I., Pourtein, M., Widjenes, J., Ticchioni, M. and Bernard, A. (2000). CD99 (E2) up-regulates α4β1-dependent T cell adhesion to inflamed vascular endothelium under flow conditions. Eur J Immunol 30(10): 3061-3065.
  2. Franciszkiewicz, K., Le Floc'h, A., Boutet, M., Vergnon, I., Schmitt, A. and Mami-Chouaib, F. (2013). CD103 or LFA-1 engagement at the immune synapse between cytotoxic T cells and tumor cells promotes maturation and regulates T-cell effector functions. Cancer Res 73(2): 617-628. 
  3. Rosenthal-Allieri, M. A., Ticchioni, M., Breittmayer, J. P., Shimizu, Y. and Bernard, A. (2005). Influence of β1 integrin intracytoplasmic domains in the regulation of VLA-4-mediated adhesion of human T cells to VCAM-1 under flow conditions. J Immunol 175(2): 1214-1223.

简介

该方法允许使用流动室中的剪切力评估T淋巴细胞和特异性粘附靶细胞之间的相对粘附强度。 其基于在微流体系统中T细胞和粘附的肿瘤细胞之间形成的缀合物对剪切应力的抗性的测量。 为此,将用CellTracker探针染色的T细胞加入含有单层粘附靶细胞的流动通道中,然后使用荧光显微镜记录在恒定剪切应力下的其逐渐分离。

材料和试剂

  1. 粘附的肿瘤细胞[例如非小细胞肺癌(NSCLC)细胞系]
  2. 特异性T细胞克隆(由自体肿瘤浸润性T淋巴细胞(TIL)或外周血淋巴细胞(PBL)产生)
  3. RPMI 1640(Life Technologies,Gibco ,目录号:61870044)
  4. DMEM-F12(Life Technologies,Gibco ,目录号:31331093)
  5. UltroserG(Pall,目录号:15950-017)
  6. 胎牛血清(Life Technologies,Gibco ,目录号:10270-106)
  7. 人血清AB(Institut de Biotechnologies Jacques Boy)
  8. 青霉素和链霉素(Life Technologies,Gibco ,目录号:15140122)
  9. 丙酮酸钠(Life Technologies,Gibco ,目录号11360029)
  10. IL-2
  11. 10x PBS(Life Technologies,Gibco ,目录号:70011-036)
  12. CellTracker探针(CellTracker TM Green CMFDA)(Life Technologies,Invitrogen TM,目录号:C2925)
  13. 完全DMEM:肿瘤细胞培养基(LC培养基)(参见配方)
  14. 基于RPMI的T细胞完全培养基(参见配方)

设备

  1. 具有加热孵育室和CO 2供应的显微镜Zeiss LSM-510(ZEISS)
  2. Micro-Slides VI,ibiTreat(ibidi GmbH,目录号:80606),两个具有塑料夹的硅管(1.6mm内径),两个Elbow Luer连接器(ibidi GmbH,目录号:80646)
  3. 注射泵(高流速> 50ml/min)
  4. 60ml注射器(Becton,Dickinson and Company,目录号:300866)
  5. 在37℃,5%CO 2/h的湿化培养箱中
  6. 废物流缓冲区(Erlenmeyer)的收件人
  7. 离心机(Beckman Coulter,型号:GS-6R)

程序

  1. 粘附肿瘤细胞制备
    1. 通过在LC培养基中加入60μl肿瘤细胞悬浮液将种子贴壁肿瘤细胞转入IBIDI通道。 微滑动VI,ibiTreat的特点如下:
      通道数:6
      每个通道最小容量:30微升
      通道长度:17毫米
      通道宽度:3.8毫米
      通道高度:0.4 mm
      生长区域:每个通道有0.6厘米 2
      肿瘤细胞浓度可根据细胞类型(例如,对于参考文献1中描述的NSCLC细胞系为1.6×10 6个细胞/ml)而变化。 在实验当天,细胞应达到90-95%的汇合
    2. 孵育IBIDI幻灯片在潮湿的孵化器在37℃至少2小时细胞附着。
    3. 轻轻地用另一个60微升的液体培养基填充水库。 避免直接移液到通道中,以免分离细胞
    4. 在37℃,5%CO 2孵育过夜。
      注意:在肿瘤细胞治疗(例如siRNA转染)的情况下,应该在使用相同的实验条件之前将细胞铺板两天。 中等可能需要每24小时更换一次。 确保细胞都存活,并且在实验当天达到90-95%汇合。

  2. T细胞制备
    1. 用PBS 1x洗涤T细胞,在350×g离心5分钟
    2. 根据制造商的方案用CellTracker Green(CMFDA)染色细胞。 简言之,将细胞以2×10 6个/ml的浓度重悬于PBS中,并加入一个体积的稀释在PBS(终浓度1μM)中的CellTracker Green(CMFDA 2x)。 在37℃下孵育15分钟。
    3. 通过在350×g离心5分钟,用基于RPMI的完全培养基洗涤T细胞两次。
    4. 在T细胞培养基中重悬T细胞,最终浓度为2×10 6个细胞/ml,在24个平底孔板中。
    5. 在37℃,5%CO 2的潮湿培养箱中培养T细胞过夜。

  3. 流动条件下的T细胞粘附强度
    1. 第二天,将显微镜的恒温室在37℃和5%CO 2(图1A)温暖。
    2. 在37℃和5%CO 2下,在培养箱内平衡基于RPMI的完全培养基(500ml)。
    3. 准备注射泵(图1B)。
    4. 将带有塑料夹子(位置关闭)的管连接到注射器,并用预热介质填充注射器。放入Elbow Luer连接器并灌注管(图1A)。
    5. 将IBIDI载玻片(图1C)放在显微镜物镜(20x)下(图1D)。确保油箱完全充满。如果没有,请添加一些介质。
    6. 将管连接到IBIDI通道的一个末端,确保内部没有残留气泡。这一步是关键,因为气泡增加了肿瘤细胞脱离的风险,影响流速,甚至可以停止流动。
                  


      图1.流系统。 A。全流系统; B.泵系统; C.微滑块的细节IBIDI连接; D.在采集期间,载玻片在显微镜下固定并连接到泵系统。

    7. 使用第二个管将通道的相对末端与瓶子收集废物连接(图2)。


      红色:粘附肿瘤细胞层
      绿色:T细胞
      蓝色:LC介质
      白色箭头:流向
      图2.流动条件下的T细胞粘附。 将染色的T淋巴细胞在先前接种到IBIDI通道中的单层自体肿瘤细胞上孵育15分钟。 IBIDI载玻片然后通过硅管在一侧连接到泵(注射器装有预热介质),另一侧连接到废物接收器。

    8. 释放将注射器连接到IBIDI载玻片的管上的夹子(图1A)。
      注意:测试系统以验证不分离肿瘤细胞的最大流速。该比率将用于确定肿瘤细胞上的T细胞粘附强度。
    9. 通过在350×g离心5分钟用RPMI洗涤T细胞
    10. 在RPMI培养基中重悬T细胞,最终浓度为2×10 6个细胞/ml。
    11. 用50μlT细胞悬浮液替换填充通道的培养基。 小心不要分离肿瘤细胞或在通道内引入气泡。
    12. 在37℃孵育15分钟。
    13. 孵育后,使用与测试分析相同的条件制备流动系统
    14. 在注射器内加入50 ml预热的培养基
    15. 在流动之前开始采集(图1D)。
    16. 每2秒采集一次图像,持续60秒。 预期T细胞比不表达这些分子的肿瘤细胞更牢固地粘附于表达粘附分子(例如由T细胞表达的整联蛋白的配体)的肿瘤细胞。 (图3)


      图3.在T细胞粘附方案期间在不同时间采集的代表性图像。 A。 0秒; B. 250秒; C.流速为100ml/h时为640秒

食谱

  1. 完全DMEM:肿瘤细胞培养基(LC)
    DMEM-F12补充:
    10%补充胎牛血清
    1%UltroserG
    1%青霉素和链霉素 1%丙酮酸钠
  2. 基于RPMI的T细胞完全培养基
    RPMI 1640,补充有10%人血清AB
    1%青霉素和链霉素 1%丙酮酸钠 IL-2(100U/ml)

致谢

我们感谢SophieSalomé-Desmoulez对共聚焦显微镜的帮助。 这项工作得到了INSERM,癌症研究协会(ARC),国家癌症研究所(INCa),癌症研究所和法兰西岛大学(IDF)的资助。 MB是来自CancéropôleIDF的奖学金的接受者。

参考文献

  1. Bernard,G.,Raimondi,V.,Alberti,I.,Pourtein,M.,Widjenes,J.,Ticchioni,M.and Bernard,A。(2000)。 CD99(E2)在流动条件下上调α4β1依赖性T细胞粘附至发炎的血管内皮。 Eur J Immunol 30(10):3061-3065。
  2. Franciszkiewicz,K.,Le Floc'h,A.,Boutet,M.,Vergnon,I.,Schmitt,A.和Mami-Chouaib,F.(2013)。 CD103或LFA-1参与细胞毒性T细胞和肿瘤细胞之间的免疫突触促进成熟和调节T-cell effector functions。 Cancer Res 73(2):617-628。 
  3. Rosenthal-Allieri,M.A.,Ticchioni,M.,Breittmayer,J.P.,Shimizu,Y。和Bernard,A。(2005)。 β1整合素胞质内结构域在VLA-4介导的人类T细胞粘附的调节中的影响VCAM-1在流动条件下。 175(2):1214-1223。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Boutet, M., Franciszkiewicz, K., Floc’h, A. L. and Mami-Chouaib, F. (2013). Protocol for T-cell Adhesion Strength on Tumor Cells under Flow Conditions. Bio-protocol 3(20): e936. DOI: 10.21769/BioProtoc.936.
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