Volume 49 Number 3

Efficient purification of mouse Treg cells based on quantitative imaging full-spectrum flow cytometry

Yingying Huang^a, Xiaoyu Meng^b, Dan Yang^a, Yanwei Li^a, Jiajia Wang^a, Xin Shen^a, Shaoyun Chen^c,*

 
ABSTRACT:     AnaccurateandefficientmethodfortheisolationandpurificationofRegulatoryT(Treg)cellsfromprimary mouse tissues was established based on the quantitative imaging full-spectrum flow cell sorter (BD FACSDiscover S8). The differences between this method and the traditional flow cell sorter (Aria II) were comprehensively evaluated. Additionally, the effects of different centrifugation conditions on the sorted cells were tested. The results demonstrated that the efficiency of S8, exceeding 94%, significantly outperforming Aria II. Under the conditions of EP (1.5 ml tubes centrifuged directly in the Eppendorf centrifuge) and 15EP(1.5mlcentrifugaltubemountedona15mlcentrifugaltube and the samples centrifuged in a low-speed centrifuge), the yield after S8 sorting was not significantly different from that of Aria II, yet the viability of the former was superior. For samples sorted using either Aria II or S8, centrifugation under 15 EP conditions resulted in a lower cell apoptosis rate. The morphology of samples sorted by Aria II required microscope observation, which could not be used for objective quantitative analysis and had a low flux. In contrast, samples sorted by S8 could simultaneously captured cell morphology imaging while recording flow cytometry data, being fast and capable of analyzing the position and content of the target protein in the cell. This new method has high sorting efficiency, excellent viability, and a real-time image interaction function, which is applicable to the sorting of precious rare samples.

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* Corresponding author, E-mail: csy@zcmu.edu.cn

Received 2 Jan 2025, Accepted 0 0000