Isolation and detection of T follicular helper cells in human peripheral blood

Abstract:

Objective：To explore a method to isolate and detect T follicular helper（Tfh） cells. Methods：Tfh cell in human peripheral blood was isolated by density gradient centrifugation（DGC） plus magnetic-activated cell sorting（MACS） plus flow cytometric sorting（FCS）. Cell purity and cell activity were detected by flow cytometry（FCM）；relative expressions of Bcl6 mRNA by real-time PCR；expressions，location and morphology of cell surface molecules CD4 and CXCR5 by confocal laser scanning microscopy，function by FCM. Results：①After sorting，purity of Tfh cell（87.69±5.01）%，was significantly enhanced before sorting（P<0.05） and cell activity was （98.21±0.85）%. ②Expressions of special transcription factor of Tfh cells-Bcl6 were significantly increased after sorting com－pared with those before sorting（P<0.05）；after sorting，the purity of the cell was high. ③Morphology of Tfh cell was complete；green fluorescence of CD4 and CXCR5 was expressed in cell membrane and can be fused. ④Expressions of Tfh cell function related cell sur－face molecules PD-1，ICOS and CD40L can be dected by FCM. Conclusions：Tfh cell with high purity and activity can be sorted through ways of density gradient centrifugation plus MACS plus FCM，then the function of them can be tested by FCM.