PBMCs were isolated from frozen leukapheresis
samples (Stemcell Technologies) or whole blood samples. Venous blood
samples were collected in EDTA tubes and supplemented with a 15×
volume of cold (4 °C) isotonic ammonium chloride solution, mixed
by inversion at room temperature for 10 min using a rotary mixer set
to ∼500 rpm to allow RBC lysis, and then centrifuged at 250g for 10 min. Cell pellets were then resuspended in 1 mL
of PBS, and this cell suspension was layered over 10 mL of Ficoll-Paque
PLUS media (Cytiva 17144002) in a 15 mL centrifuge tube and centrifuged
at 500g for 20 min in a swinging buck rotor to isolate
PBMCs following the manufacturer’s instructions. Isolated PBMCs
were resuspended in 5 mL of AIM V cell culture media (Fisher Scientific
31-035-025); aliquots were analyzed to determine viable cell concentrations
by staining cells with a 0.4% Trypan Blue solution, and cell suspensions
were adjusted to a final concentration of 3 × 106/mL
in AIM V cell culture media (Fisher Scientific 31-035-025), mixed
with 40% fetal bovine serum and 20% dimethyl sulfoxide, and then stored
in the vapor phase of a liquid nitrogen tank.
samples (Stemcell Technologies) or whole blood samples. Venous blood
samples were collected in EDTA tubes and supplemented with a 15×
volume of cold (4 °C) isotonic ammonium chloride solution, mixed
by inversion at room temperature for 10 min using a rotary mixer set
to ∼500 rpm to allow RBC lysis, and then centrifuged at 250g for 10 min. Cell pellets were then resuspended in 1 mL
of PBS, and this cell suspension was layered over 10 mL of Ficoll-Paque
PLUS media (Cytiva 17144002) in a 15 mL centrifuge tube and centrifuged
at 500g for 20 min in a swinging buck rotor to isolate
PBMCs following the manufacturer’s instructions. Isolated PBMCs
were resuspended in 5 mL of AIM V cell culture media (Fisher Scientific
31-035-025); aliquots were analyzed to determine viable cell concentrations
by staining cells with a 0.4% Trypan Blue solution, and cell suspensions
were adjusted to a final concentration of 3 × 106/mL
in AIM V cell culture media (Fisher Scientific 31-035-025), mixed
with 40% fetal bovine serum and 20% dimethyl sulfoxide, and then stored
in the vapor phase of a liquid nitrogen tank.