Cd56 conjugated microbeads

F10 nutrient mixture (HAM), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum (HS), penicillin/streptomycin (P/S), and Fungizone antimycotic (FZ) were obtained from Invitrogen (Taastrup, Denmark). CD56-conjugated microbeads were from Miltenyi Biotec (Lund, Sweden). C1^TM System and C1 integrated fluidic circuits (IFCs) were from Fluidigm (San Francisco, CA, United States). LIVE/DEAD cell staining solution, ActinRed and NucBlue ReadyProbes antibodies, and Megaplex PreAmp primers were from Thermo Scientific (Taastrup, Denmark).

The CIK cells were washed twice with PBS buffer containing BSA (0.5%) and EDTA (2 mM), incubated with CD56-conjugated microbeads (Miltenyi Biotec, Auburn, CA), and passed through a LS+ magnetic-activated cell sorting (MACS) separation column, according to the manufacturer’s instructions (Miltenyi). The eluant contained the CD3+ CD56− subset, while the positively selected CD56+ cells were collected by flushing the column. Because CIK cells usually contain a small proportion of CD3−CD56+ NK cells, which varies depending on the donor, CIK cells with a low proportion of NK cells were selected to ensure that the purity of CD3+CD56+ cells after sorting was greater than 95% to avoid the influence of NK cells.