分选方式对细胞能量代谢的影响研究  

Study on the Effect of Different Sorting Methods on Cell Energy Metabolism

作　　者：邢月婷 李艳伟[2] 黄莹莹[2] 王佳佳[2] 郭春 沈鑫 周南 沈红英 宋兴辉 XING Yueting;LI Yanwei;HUANG Yingying;WANG Jiajia;GUO Chun;SHEN Xin;ZHOU Nan;SHEN Hongying;SONG Xinghui(Women’s Hospital School of Medicine,Zhejiang University,Hangzhou 310006,China;Core Facilities,School of Medicine,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学医学院附属妇产科医院,杭州310006 [2]浙江大学医学院公共技术平台,杭州310058

出　　处：《中国细胞生物学学报》2024年第5期964-975,共12页Chinese Journal of Cell Biology

基　　金：浙江省教育厅一般科研项目(批准号:Y202249909)资助的课题。

摘　　要：Seahorse XF(extracellular flux)是从能量的角度检测代谢及调控的方法,因其实时检测的优点,具有非常好的应用前景,但该技术要求目的细胞纯度高、活性好,因此需要对原代细胞的分选条件进行优化。该文通过免疫磁珠分选技术和流式细胞分选技术对小鼠脾脏CD4+T细胞进行研究,结果发现两种分选方式纯化的CD4+T细胞在活性氧水平上无明显差异,相较于磁珠分选,流式分选纯化的CD4+T细胞线粒体膜电位、ATP合成相关的氧耗值和线粒体储备呼吸能力均呈现下降趋势,说明磁珠分选相较于流式分选对CD4+T细胞能量代谢的损伤更小。为了进一步了解流式分选的影响,该文对分选条件进行了研究。通过Moflo AstriosEQ分选仪中不加电分选模式(SD),加电分选模式不同偏振电压(700 V、2400 V、5000 V)以及同一偏振电压下不同液滴加电(35%、70%)对HEK 293T和Jurkat进行分选,结果发现SD组相较于加电分选模式对细胞代谢干扰较小,加电分选模式中5000 V实验组对细胞代谢有显著影响。同一偏振电压条件下,不同液滴加电实验组均未对细胞产生显著干扰。综上,磁珠法更适用于小鼠原代CD4+T细胞能量代谢实验前分选预处理。流式法用于能量代谢实验预处理时建议优先选择不加电分选模式,选择加电分选模式时建议使用小于2400 V的偏振电压。该实验结果为能量代谢检测实验提供了分选方式和分选条件的数据参考。Seahorse extracellular flux technology is used to detect metabolism and its regulation from an energy perspective.It has good application prospects due to its advantages such as fewer cells and real-time detection.Optimizing sorting parameters is essential to ensure the isolation of high-purity and high-activity cells.Taking CD4+T primary cells from mouse spleen as an example,it was shown that there were no significant differences in reactive oxygen species between MACS(magnetic activated cell sorting)and FACS(fluorescence-activated cell sorting).However,all parameters including mitochondrial membrane potential,ATP production and spare respiratory capacity of CD4+T cells from FACS showed the down-warded trend when compared with CD4+T cells from MACS.This indicated that MACS caused less damage to CD4+T cells in terms of cell energy metabolism compared with FACS.To investigate the impact of FACS,this study delved into sorting parameters.HEK 293T cells and Jurkat cells underwent sorting using both straight down sorting,varying voltages(700 V,2400 V,5000 V)and stream deflecions(35%,70%)in the Moflo AstriosEQ sorter.The findings revealed that straight down sorting had a lesser impact on cell metabolism compared to sorting at different voltages.At 5000 V,the cell metabolism exhibited a significant difference compared to both the 700 V and 2400 V groups.Different stream deflections had no significant interference to the cells under the same plate voltages.Thus,MACS is suitable for conducting energy metabolism experiments with mouse primary CD4+T cells,while straight down sorting is preferable for FACS experiments focusing on energy metabolism.If sorting by charge mode is necessary,it is advisable to use a voltage value lower than 2400 V.The results of this experiment provide a data set as a reference of sorting conditions in energy metabolism experiment.

关 键 词：磁珠分选 流式分选 活性氧 线粒体膜电位 能量代谢 

分 类 号：Q251[生物学—细胞生物学]