流式细胞术检测NK细胞的细胞毒作用的两种方法比较
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摘要
目的:比较并优化流式细胞术检测人NK细胞体外细胞毒功能的方法。方法:采用Calcein-AM释放法或CFSE/7-AAD法分别对靶细胞进行标记,按10∶1和20∶1效靶比与7例NK细胞在37℃5%CO2培养箱共培养4 h,流式细胞术测定两种不同标记方法的靶细胞死亡率。结果:各实验组中CFSE/7-AAD法检出的靶细胞死亡率均高于Calcein-AM释放法,在低效靶比、弱细胞毒作用条件下,差异具有统计学意义。细胞毒作用较弱时,Calcein-AM释放法平均荧强度变化不显著、结果误差较大;而CFSE/7-AAD法则能更加敏感地检出靶细胞的死亡率。结论:CFSE/7-AAD法能够特异、灵敏地检出NK细胞的细胞毒活性,所获得的结果更为可靠。
Objective: To compare and optimize a method for detecting cytotoxicity mediated by vitro expanded NK cells using flow cytometry. Methods: High purity NK cells were applied as effector cells. Calcein-AM release or CFSE/7-AAD was used to label target cells respectively. Seven different NK cells and different target cells were co-incubated at 37℃ 5% CO2 incubator for 4 hours with a E ∶T ratio of 10 ∶1 or 20 ∶1,respectively. The death rate of target cells was detected by flow cytometry. Results: Comparing the two different staining method,CFSE/7-AAD double staining method showed better capability than Calcein-AM release in detecting death rate of target cells,particularly in the condition of lower E ∶T ratios and weak cytotoxicity. Under situation of weak cytotoxicity,the mean fluorescence intensity( MFI) of target cells labeled by Calcein-AM release did not show significant alteration and may result in measurement error in calculating the death rate of target cells. Conclusion: CFSE/7-AAD double staining method exhibits more sensitive in detecting cytotoxicity mediated by NK cells and the obtained results are more reliable.
Objective: To compare and optimize a method for detecting cytotoxicity mediated by vitro expanded NK cells using flow cytometry. Methods: High purity NK cells were applied as effector cells. Calcein-AM release or CFSE/7-AAD was used to label target cells respectively. Seven different NK cells and different target cells were co-incubated at 37℃ 5% CO2 incubator for 4 hours with a E ∶T ratio of 10 ∶1 or 20 ∶1,respectively. The death rate of target cells was detected by flow cytometry. Results: Comparing the two different staining method,CFSE/7-AAD double staining method showed better capability than Calcein-AM release in detecting death rate of target cells,particularly in the condition of lower E ∶T ratios and weak cytotoxicity. Under situation of weak cytotoxicity,the mean fluorescence intensity( MFI) of target cells labeled by Calcein-AM release did not show significant alteration and may result in measurement error in calculating the death rate of target cells. Conclusion: CFSE/7-AAD double staining method exhibits more sensitive in detecting cytotoxicity mediated by NK cells and the obtained results are more reliable.
引文
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[16] Zhang J,Wang Y,Wu L,et al. Application of an improved flow cytometry-based NK cell activity assay in adult hemophagocytic lymphohistiocytosis[J]. Int J Hematol,2017,105(6):828-834.
[17]张嘉,王晶石,王旖旎,等.一种新型稳定的流式细胞术检测NK细胞活性的方法[J].白血病·淋巴瘤,2015,24(8):464-466.Zhang J,Wang JS,Wang YN,et al. A new stable detection method of natural killer cell activity by flow cytometry[J]. J Leuk Lymph,2015,24(8):464-466.
[18] Ozdemir O,Ravindranath Y,Savasan S,et al. Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three-color flow cytometry[J]. Cytometry A,2003,56(1):53-60.
[2]徐彤,田志刚,张彩,等.人外周血NK细胞纯化方法的初步比较[J].中国免疫学杂志,2000,16(11):594-597.Xu T,Tian ZG,Zhang C,et al. Preliminary study on the purification method of human NK cell[J]. Chin J Immunol,2000,16(11):594-597.
[3]王新利,李卿,高婷,等.一种经济高效的人外周血NK细胞纯化方法的建立[J].现代肿瘤医学,2010,18(3):437-439.Wang XL,Li Q,Gao T,et al. Establishment of an economical method to obtain NK cells with high purity[J]. J Mod Oncol,2010,18(3):437-439.
[4]李晓红,马健,吴晓雄,等.细胞因子联合高效扩增高纯度人外周血来源NK细胞并观察其细胞功能的变化[J].中华血液学杂志,2009,30(6):404-408.Li XH,Ma J,Wu XX,et al. Study on ex vivo expansion of highly purified NK cells from human peripheral biood and changes in their function[J]. Chin J Hematol,2009,30(6):404-408.
[5] CholujováD,JakubíkováJ,Kubes M,et al. Comparative study of four fluorescent probes for evaluation of natural killer cell cytotoxicity assays[J]. Immunobiology,2008,213(8):629-640.
[6] Jang YY,Cho D,Kim SK,et al. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells[J]. Ann Clin Lab Sci,2012,42(1):42-49.
[7] Rossignol A,Bonnaudet V,Clémenceau B,et al. A highperformance, non-radioactive potency assay for measuring cytotoxicity:A full substitute of the chromium-release assay targeting the regulatory-compliance objective[J]. Mabs,2017,9(3):521-535.
[8]郭继强,韩亚萍,李芳,等.流式细胞术三种染色方法检测体外纯化扩增的NK细胞的细胞毒作用比较[J].中国实验血液学杂志,2016,24(6):1691-1697.Guo JQ,Han YP,Li F,et al. Comparison of ex vivo expanded and highly purified NK cell-mediated cytotoxicity detected by 3different staining methods of flow cytometry[J]. J Exp Hematol,2016,24(6):1694-1697.
[9] Jin Q,Jiang L,Chen Q,et al. Rapid flow cytometry-based assay for the evaluation of gammadelta T cell-mediated cytotoxicity[J]. Mol Med Rep,2018,17(3):3555-3562.
[10] Toth G,Szollosi J,Vereb G,et al. Quantitating ADCC against adherent cells:Impedance-based detection is superior to release,membrane permeability,or caspase activation assays in resolving antibody dose response[J]. Cytometry A,2017,91(10):1021-1029.
[11] Jedema I,Van RM,Willemze R,et al. New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population[J].Blood,2004,103(7):2677-2682.
[12] Ivanova OK,Sharapova TN,Romanova EA,et al. CD3+CD8+NKG2D+T lymphocytes induce apoptosis and necroptosis in HLA-negative cells via Fas L-fas interaction[J]. J Cell Biochem,2017,118(10):3359-3366.
[13] Konjevic G,Vuletic A,Mirjacic Martinovic K,et al. Evaluation of the functional capacity of NK cells of melanoma patients in an in vitro model of NK cell contact with K562 and femx tumor cell lines[J]. J Membr Biol,2017,250(5):507-516.
[14] Christian Demanet,Arend Mulder,Veronique Deneys,et al.Down-regulation of HLA-A and HLA-Bw6,but not HLA-Bw4,allospecificities in leukemic cells:an escape mechanism from CTL and NK attack?[J]. Blood,2004,103(8):3122-3130;
[15] Reusing SB,Manser AR, Enczmann J, et al. Selective downregulation of HLA-C and HLA-E in childhood acute lymphoblastic leukaemia[J]. Br J Haematol,2016,174(3):477-480.
[16] Zhang J,Wang Y,Wu L,et al. Application of an improved flow cytometry-based NK cell activity assay in adult hemophagocytic lymphohistiocytosis[J]. Int J Hematol,2017,105(6):828-834.
[17]张嘉,王晶石,王旖旎,等.一种新型稳定的流式细胞术检测NK细胞活性的方法[J].白血病·淋巴瘤,2015,24(8):464-466.Zhang J,Wang JS,Wang YN,et al. A new stable detection method of natural killer cell activity by flow cytometry[J]. J Leuk Lymph,2015,24(8):464-466.
[18] Ozdemir O,Ravindranath Y,Savasan S,et al. Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three-color flow cytometry[J]. Cytometry A,2003,56(1):53-60.