Single-cell suspensions of PBMC preparation
Density gradient centrifugation method was utilized to obtain PBMCs. We
used PBS (Solarbio, P1022-500) to dilute the whole blood sample at a
ratio of 1:1, and then slowly added the diluted whole blood sample to
approximately 2/3 of the volume of Ficoll (GE Healthcare, 17-1440-02).
After centrifugation at 400 g for 35 min, three layers were
obtained based on size and density. The middle cell suspension layer was
taken into a new 15-mL centrifuge tube and was filled with PBS and
centrifuged again at 300 g for 7 min, supernatant was discarded
and the pellet containing PBMCs were washed twice, and the final sample
was resuspended in PBS to obtain a final concentration of
1×105 cells/mL.. Viability staining with 0.4% Trypan
blue solution (Sigma, T8154) was performed and viable cells were counted
under the microscope.