Ficoll paque premium

Fasting blood samples were collected in EDTA tubes and centrifuged to collect plasma. Plasma was directly frozen (−80 °C). PBMCs were isolated from the remaining blood using Ficoll-Paque™ PREMIUM (VWR) and immediately frozen at −80 °C.

Whole blood was collected from healthy donors in citrate tubes according to an approved institutional standard operating procedure. PBMCs were purified using gradient centrifugation through Ficoll-Paque Premium (1.078 g/mL density, VWR, Lutterworth, Leicestershire, England) in Greiner Bio-One Leucosep tubes (Cat # 227290P, Greiner Bio-One North America Inc., Monroe, NC) as described by the manufacturer (45 (link)). Briefly, 50 mL of blood per donor was collected in citrated tubes by the USAISR Research Blood Bank; PBMCs were separated from plasma and red blood cells and washed twice with Dulbecco’s phosphate buffered saline (dPBS, Gibco #14190-144, without calcium or magnesium chloride). Both single donor (hSD-PBMC) and pooled donor (hPD-PBMC, 8–10 donors) PBMC preparations were prepared. PBMCs were resuspended at 1–2 × 10⁷ per mL in CryoStor10, frozen at 1°C/min cooling rate in a Nalgene™ Mr Frosty and stored in liquid nitrogen until use. Viability upon thaw was 94 ± 2.4% (n = 9) by 1:1 dilution with acridine orange propidium iodide (AOPI) stain and cell counting in the Cellaca MX High-Throughput Automated Cell Counter (Nexcelom Bioscience, Lawrence, MA).