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Standard 4-hours Chromium-51 (51Cr) Release Assay
标准化的4小时铬-51(51Cr)的释放试验   

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Abstract

51Cr-release assays are commonly used for the precise and accurate quantification of cytotoxicity, particularly in the study of tumor cytolysis. This test has the advantage of requiring only very few cells.

Materials and Reagents

  1. Target cells: Acute Myeloid Leukemia (AML) cell line (U937 or HL60 from ATCC) or AML cells isolated from patients with AML
  2. Effector cells: γδ T cells isolated from Healthy volunteer provided by Etablissement
  3. Francais du Sang (EFS) or patients
  4. Medium : RPMI 1640 (Life Technologies, Gibco®)
  5. Fetal Calf Serum (FCS) heat-inactivated 1 h at 56 °C
  6. Chromium-51 (51Cr, 5 mCi/ml) (PerkinElmer)
  7. Complete medium = RPMI 1640 medium supplemented with 10% heat-inactivated FCS

Equipment

  1. Centrifuge for microplates
  2. Microplates (96 well round-bottom) for cell incubations
  3. LumaPlateTM (PerkinElmer)
  4. Liquid scintillation counter
  5. Incubator

Controls

  1. Spontaneous Release: Target cells were incubated alone (replace effector cells by 50 μl of media). After 4 h of incubation, wells were centrifuged and 50 μl of supernatant were recovered.
  2. Maximum load: Target cells were incubated alone (replace effector cells by 50 μl of media). After 4 h of incubation, wells were mixed and 50 μl of cell suspension were recovered rather than disrupt the cell membrane to release the radioactivity into the supernatant). This avoids the use of detergent.

Procedure

  1. 51Cr labeling the target cells
    1. Wash targets (2.106 per condition) in 15 ml of medium.
    2. Centrifuge targets; discard the supernatant and resuspend pellets in 20 μl of FCS.
    3. Add 100 μCi of 51Cr (20 μl of stock solution at 5 mCi/ml) for one hour in an incubator at 37 °C.
      NB: To define the dose of chromium necessary to mark a cell type, it must be ensured that the spontaneous release is less than 10% of maximum load.
    4. Wash cells and resuspend in 60,000 cells per ml of complete medium.
    5. Add 3,000 Target cells per well in 50 μl of a round-bottom 96 well plate.

  2. Incubation of target cells with effector cells
    1. Add effector cells (50 μl) at E:T (Effetor:Target) ratios of 30:1, 20:1, 10:1, and 1:1. In parallel, set up controls (Spontaneous Release, Maximum load) and incubate 4 h at 37 °C.

  3. Analysis
    1. Centrifuge samples and collect supernatant (50 μl).
    2. Supernatants are dried on a LumaPlateTM (PerkinElmer) overnight and counted in a liquid scintillation counter.
      The percentage of specific lysis was calculated using the standard formula [(experimental - spontaneous release)/(maximum load- spontaneous release) x 100] and expressed as the mean of triplicate samples

Acknowledgments

This protocol is adapted from Gertner-Dardenne et al. (2012).

References

  1. Gertner-Dardenne, J., Castellano, R., Mamessier, E., Garbit, S., Kochbati, E., Etienne, A., Charbonnier, A., Collette, Y., Vey, N. and Olive, D. (2012). Human Vgamma9Vdelta2 T cells specifically recognize and kill acute myeloid leukemic blasts. J Immunol 188(9): 4701-4708.

简介

通常使用Cr释放测定来精确和准确地定量细胞毒性,特别是在肿瘤细胞溶解的研究中。 该测试具有仅需要非常少的电池的优点。

材料和试剂

  1. 靶细胞:急性骨髓性白血病(AML)细胞系(来自ATCC的U937或HL60)或从AML患者分离的AML细胞
  2. 效应细胞:从由Etablissement提供的健康志愿者分离的γδT细胞
  3. Francais du Sang(EFS)或病人
  4. 介质:RPMI 1640(Life Technologies,Gibco )
  5. 胎牛血清(FCS)在56°C热灭活1小时
  6. 铬-51(51 Cr,5mCi/ml)(PerkinElmer)
  7. 完全培养基=补充有10%热灭活的FCS的RPMI 1640培养基

设备

  1. 微量离心机
  2. 微孔板(96孔圆底)用于细胞培养
  3. LumaPlate TM (PerkinElmer)
  4. 液体闪烁计数器
  5. 孵化器

控件

  1. 自发释放:单独孵育靶细胞(用50μl培养基替代效应细胞)。 孵育4小时后,离心孔,回收50μl上清液
  2. 最大负载:靶细胞单独孵育(用50μl培养基替代效应细胞)。 孵育4小时后,将孔混合,回收50μl细胞悬浮液,而不是破坏细胞膜以释放放射性到上清液中。 这避免使用洗涤剂。

程序

  1. 51 Cr标记靶细胞
    1. 在15 ml培养基中洗涤目标(每种条件2.106)。
    2. 离心机目标; 弃去上清液并将沉淀重悬于20μlFCS中
    3. 在37℃的培养箱中加入100μCi的51 Cr(20μl储备液,5mCi/ml)1小时。
      注意:要定义标记细胞类型所需的铬剂量,必须确保自发释放小于最大负荷的10%。
    4. 洗涤细胞并重悬于60,000个细胞/ml完全培养基中
    5. 每孔加入3,000个靶细胞于50μl圆底96孔板中

  2. 靶细胞与效应细胞的孵育
    1. 以E:T(Effetor:Target)比例为30:1,20:1,10:1和1:1加入效应细胞(50μl)。 同时,设置控件(自发释放,最大负载),并在37°C孵育4小时。

  3. 分析
    1. 离心样品并收集上清液(50μl)
    2. 将上清液在LumaPlate TM(PerkinElmer)上干燥过夜,并在液体闪烁计数器中计数。
      使用标准公式[(实验 - 自发释放)/(最大负荷 - 自发释放)×100]计算特异性裂解的百分比,并表示为三份样品的平均值

致谢

该协议改编自Gertner-Dardenne等人(2012)。

参考文献

  1. Gertner-Dardenne,J.,Castellano,R.,Mamessier,E.,Garbit,S.,Kochbati,E.,Etienne,A.,Charbonnier,A.,Collette,Y.,Vey,N.and Olive,D 。(2012)。 人类Vgamma9Vdelta2 T细胞特异性识别和杀死急性骨髓性白血病母细胞。 J Immunol 188(9):4701-4708。
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引用:Gertner-dardenne, J. (2012). Standard 4-hours Chromium-51 (51Cr) Release Assay. Bio-protocol 2(23): e301. DOI: 10.21769/BioProtoc.301.
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