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[Bio101] Naïve T Lymphocyte Infection by Murine Stem Cell Virus (MSCV) Protocol
[Bio101] 幼稚T淋巴细胞的小鼠干细胞病毒(MSCV)感染实验方法

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Abstract

This assay can be used for studying genes related to lymphocyte proliferation and differentiation in vitro. Stimulating lymphocytes to proliferate is important for the infection efficiency of MSCV. After overnight culture, if naïve cells proliferate extensively, the following spin infection will have high efficiency. After infection, cells are ready for the following assay as they will be undergoing proliferation or differentiation.

Materials and Reagents

  1. Mouse
  2. Fetal bovine serum (FBS)
  3. Anti-CD3 and Anti-CD28 antibody (Ab) (Biolegend, catalog number: 100302 , 102102
  4. IL-2 (R&D systems, catalog number: 402-ML-020 )
  5. Polybrene (Hexadimethrine bromide) (Sigma-Aldrich, catalog number: H9268 )
  6. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
  7. Penicillin/streptomycin solution (Life Technologies, Gibco®, catalog number: 15140-122 )
  8. 2-mercaptoethanol (Life Technologies, InvitrogenTM, catalog number: 21985-023 )
  9. PBS solution (1x phosphate buffered saline) (Life Technologies, Gibco®, catalog number: 10010-023 )
  10. MSCV (Clontech, catalog number: 634401 )
  11. RPMI-1640 medium (Life Technologies, Gibco®, catalog number: 11875-093 ) (see Recipes)

Equipment

  1. Centrifuges
  2. 24-well plate
  3. Water bath
  4. Parafilm

Procedure

  1. Coat plate with anti-CD3 and anti-CD28 Ab overnight.
    Add 400 μl/well PBS containing 1 μg/ml of anti-CD3 and 0.5 μg/ml of anti-CD28 to 24-well plate. Place lid or seal with parafilm, and keep in dark 4 °C overnight. Next day, discard the antibodies by aspiration and add 400 μl/well of RPMI-1640 culture medium; do not let the plate dry.
  2. Sacrifice mouse and sort CD4+CD44 loCD62Lhi naïve T cells.
  3. Seed 5 x 106 cells/well for 24-well plate in PRMI-1640 culture medium and culture cells overnight.
  4. Spliting cells in the ratio 1:2 (separate one well cells into two wells), adding IL-2 at final concentration of 50 U/ml, culture for 6 h (37 °C, 5% CO2).
  5. Spin the plate at 1,200 RPM for 5 min (room temperature), remove culture medium by aspiration, and add 1 ml RPMI-1640 with proper amount of MSCV particles (virus concentration depends on the titter which is more than 106 cfu/ml) and 5-10 μg/ml of polybrene. Spin the plate at 2,500 RPM (37 °C) for 90 min.
  6. Remove supernatant and add RPMI-1640 culture medium for differentiation or proliferation.

Recipes

  1. RPMI-1640 culture medium
    Supplemented with
    5% FBS
    1% Penicillin/Streptomycin solution
    50 μM 2-mercaptoethanol

简介

该测定法可用于体外研究与淋巴细胞增殖和分化相关的基因。 刺激淋巴细胞增殖对于MSCV的感染效率是重要的。 在过夜培养后,如果初始细胞广泛增殖,以下自旋感染将具有高效率。 感染后,细胞准备用于以下测定,因为它们将经历增殖或分化。

材料和试剂

  1. 鼠标
  2. 胎牛血清(FBS)
  3. 抗CD3和抗CD28抗体(Ab)(Biolegend,目录号:100302,102102)
  4. IL-2(R& D systems,目录号:402-ML-020)
  5. 聚凝胺(Hexadimethrine bromide)(Sigma-Aldrich,目录号:H9268)
  6. 胎牛血清(FBS)(Gemini Bio-Products,目录号:900-108)
  7. 青霉素/链霉素溶液(Life Technologies,Gibco ,目录号:15140-122)
  8. 2-巯基乙醇(Life Technologies,Invitrogen TM ,目录号:21985-023)
  9. PBS溶液(1x磷酸盐缓冲盐水)(Life Technologies,Gibco ,目录号:10010-023)
  10. MSCV(Clontech,目录号:634401)
  11. RPMI-1640培养基(Life Technologies,Gibco ,目录号:11875-093)(参见Recipes)

设备

  1. 离心机
  2. 24孔板
  3. 水浴
  4. parafilm

程序

  1. 用抗CD3和抗CD28抗体涂布板过夜 向24孔板中加入400μl/孔含有1μg/ml抗CD3和0.5μg/ml抗CD28的PBS。放置盖子或密封与石蜡膜,并在暗处4℃过夜。第二天,通过抽吸弃去抗体,并加入400μl/孔的RPMI-1640培养基;不要让板干燥。
  2. 牺牲小鼠和排序CD4 + CD44 loCD62Lhi幼稚T细胞
  3. 对于24孔板在PRMI-1640培养基和培养细胞中接种5×10 6个细胞/孔过夜。
  4. 以1:2的比例分开细胞(将一个孔细胞分成两个孔),加入终浓度为50U/ml的IL-2,培养6小时(37℃,5%CO 2/>)。
  5. 在1,200RPM下旋转平板5分钟(室温),通过抽吸除去培养基,并加入1ml具有适当量的MSCV颗粒的RPMI-1640(病毒浓度取决于滴定度,大于10 6 cfu/ml)和5-10μg/ml聚凝胺。在2,500RPM(37℃)下旋转板90分钟
  6. 取出上清液,加入RPMI-1640培养基进行分化或增殖

食谱

  1. RPMI-1640培养基
    补充
    5%FBS
    1%青霉素/链霉素溶液 50μM2-巯基乙醇
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引用:Li, J. (2011). Naïve T Lymphocyte Infection by Murine Stem Cell Virus (MSCV) Protocol. Bio-protocol Bio101: e156. DOI: 10.21769/BioProtoc.156;
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