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

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, 2 400 V, 5 000 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 5 000 V, the cell metabolism exhibited a significant difference compared to both the 700 V and 2 400 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 2 400 V. The results of this experiment provide a data set as a reference of sorting conditions in energy metabolism experiment.

CSTR: 32200.14.cjcb.2024.05.0009