This unit describes several experimental protocols for inducing cytotoxic T lymphocytes and assaying their activity Remember that, in principle, any kind of cell can be used as a target cell for assaying C T L activity, but the best target cells are activated cells such as lymphoblastoid cells, tissue culture cells, or tumor cells. Author: J.E. Colligan et al. Translated by Xuitao Cao et al. This experiment is from the "Compendium of Immunology Experiments Guide".
Operation method
Cytotoxic T lymphocytes induction and detection Move Basic Option 1 Induction of cytotoxicity from C TL precursors Note: Reactive cells should be selected for in vivo sensitization based on the type of antigen. Reactive cell source: unsensitized or in vivo sensitized mouse spleen cells (see Supplementary protocols 1 and 2) Sensitization medium Stimulating cell source: unmodified or semi-antigenically modified mouse spleen cells (see Supplementary Scheme 3) R P M I -1 0 Complete Medium H B S S solubilized 0.5m g / m l mitomycin C (keep away from light) Knife bean protein A (optional): neutralized with or methyl-D-mannoside plus supernatant to cultured cells IL-2 (optional) 15 ml secondary polystyrene conical tubes with screw caps (e.g. Falcon) 2 4-well flat-bottomed microtitration plate, 2 ml volume, with lid (e.g. Costar) Note: Mice should be protected from infections that can affect the results of the experiment, such as mice and immune-suppressive viruses (e.g., Tropicana). In order to generate an in vivo response to a secondary histocompatibility antigen, a recipient should be used who is homozygous and of the same H-2 strain as the donor mouse. To generate an anti-H-Y response (a special case of the secondary antigen), skin or cells from a female recipient and a male mouse of the same strain are used. Recipient mice are transplanted with skin or intraperitoneal injections of IX IO7 to 5 X IO7 splenocytes (in a volume of 0.1 to 0.5 ml; Appendix 2E), which form an emulsion with Fuchs' Complete Adjuvant prepared by the method in Unit 1.2. Serum-free medium was used to Dilute influenza virus allantoic storage solution with 1.0 ml P B S to 300 hemagglutinin units (H A U ). Intraperitoneal injection was performed as in Appendix 2E. After in vivo immunization using the appropriate route, collect the cells and prepare a spleen single cell suspension as described for induced C T L (see Basic Option 1). It is preferable to use activated cells as target cells in 51C r release assays, whereas normal spleen cells can be used to stimulate C T L production (this avoids the time-consuming activation step). The following protocol bypasses MHC restriction and antigen specificity of CTL precursors by stimulating CTL precursors with mitogens. Reactive spleen cells were cultured in a 24-well microtitre plate with sensitization medium containing 2ug/ml of CClA. For more product details, please visit Aladdin Scientific website.
![收 集 脾 脏 细 胞 作 为 把 细 胞 的 方 法 如 下 。在 第 0 天 同 时 收 集 作 为 刺 激 细 胞 和 把 细 胞 的 脾 脏 细 胞 。将 靶 细 胞 放 在 培 养 瓶 中 拧 紧 盖 子 4°C保 存 ； 2〜 3 d 后 按 步 骤 1 中方法加入 有 丝 分 裂 原 ，拧 松 瓶 盖 后 放 CO2 培 养 箱 中 培 养 2〜 3d。 2•将细胞转移至15m l 圆锥管中，加 入 14ml R PMI-IO完全培养基，室 温 ，约 200g 离 心 5m i n 洗一遍。弃上清。 3 . 用 5ml R PMI-IO完全培养基混悬细胞。放置数分钟使细胞团块沉淀或用单层112/^m 尼龙滤网放置在15m l 圆锥管敞开的管口，过 滤 细 胞 悬 液 （用移液器枪头压下滤网形 成小的漏斗；如难以形成漏斗则改用50m l 圆锥管）。 4 . 用台盼蓝拒染法确定未沉淀细胞或滤过细胞中活细胞数（附 录 3C ; 实验所需活细胞 比例为> 8 0 % ) 。必要时采用Ficoll-Hypaque密 度 离 心 （单 元 2 . 1 ) 纯化活细胞，但 要注意，从活细胞比例低的群体中纯化的活细胞通常比从比例高的群体中纯化的活 细胞更容易发生51Cr渗漏。 5 . 在 一 个 15m l 圆锥管中离心< 5 X 106 个细胞，室温， 200g 离 心 5min。丢弃大部分上 清 ，留下细胞团块上约0.1m l 的上清。 6 . 用留下的R P M I -10完全培养基轻轻混悬细胞。加 入 0.2ml [ 少 量 即 可 （如 0.05〜 0. Iml) ] 约 lmCi/m l 51Cr溶 液 （标记肿瘤细胞和培养细胞时< 2 0 0 MCi/iLtl即可）和 2(V 1 F B S 。轻轻混匀，将培养瓶盖拧松放37°C ， 5 % C 0 2 培养箱孵育，淋巴细胞孵 育 约 45min，肿瘤细胞孵育1〜2h ，孵育期间轻轻混匀细胞可提髙标记效果。利用孵 育时间进行步骤7 操作。 7 . 按 步 骤 2〜4 制备效应细胞，用 R P M I -10完全培养基混悬至IO7 个细胞/ml。制备与 效应细胞仅存抗原特异性不同的对照细胞（未致敏细胞或无关抗原致敏细胞）。每种 效应细胞用R P M I -10完全培养基进行3 倍梯度稀释。 8 . 将 0.1m l 效 应 细 胞 （C T L 或非裂解性细胞）或对照细胞加至9 6 孔板中，每个效应细 胞 浓 度 设 3 或 4 复 孔 。用 预 期 的 C T L 活 性 计 算 不 同 的 效 应 细 胞 浓 度 ，如效靶比 (E : T )100、 20、 10、 3 (通常活化的C T L 在 E : T < 1 : 1 时即可检测到细胞裂解）。 注意，细胞浓度大于2 X I O 6 个细胞/孔通常会抑制活性。 9 . 按 步 骤 2 的方法加入14ml R P M I -10完全培养基洗涤51Cr标 记 细 胞 2 或 3 遍 ，将上清 全部吸出并收集到装放射性废物的容器中（离心中应盖上管盖）。用 R P M I -10完全 培养基混悬标记的靶细胞至IO4〜IO6 个细胞/m l 。迅速进行步骤1 0 操作。 10. 将 0.1m l 步 骤 7 制备的51C r 标 记 细 胞 （即足以产生高于背景^ lOOOcpm的细胞数） 至装有如下一种细胞的各个孔中，使终体积达到〇 .2ml/孔 ： 0. I m l 效 应 细 胞 （洗后尽快加入靶细胞） 0.1m l 对 照 淋 巴 细 胞 （步 骤 8 注解） 0.1m l 培 养 基 （检测自发51C r 释放） 11. 为促进效靶细胞间的接触，用培养板架将培养板在约200g 离 心 约 30s。 37°C 饱和湿 度 ， 5 % C O 2 培养箱中孵育3〜6h (取决于实验目的、效应细胞活力，以及靶细胞 51Cr释放的敏感性；也可以培养过夜）（室温结合， 37°C 裂解）。 12•将培养板在约200g•离心5min。将 0 •Iml Triton X -100 (裂解剂）加 入 装 有 0 •Iml 靶细胞的几个孔中，用移液器混匀后检测可释放的最大51& 。用多通道移液器每孔](http://img.dxycdn.com/trademd/upload/userfiles/image/2016/07/A14695020390072yq9qm96m9png_small.jpg)
Supplementary Scheme 1 In vivo mouse response to secondary histocompatibility antigens
Dilute cells to inject mice. Between some strains, it may be necessary to re-inject IO7 spleen cells after 10 ~Md to produce a strong in vitro re-response. After in vivo immunization by the appropriate route, prepare a single-cell suspension of spleen cells as described for induction of CTL (see Basic Option 1). For re-response, the mice providing the responding cells are usually immunized in vivo within a minimum of 1 week and a maximum of 1 year.
Adjuvant Program 4 Viral Infection of Target/Stimulated Cells Materials (see Basic Program 2 for additional materials) 
Evaluation of total CTL activity without considering antigen specificity
