Lymphosep cell separation media

PBMCs were isolated from leukocyte filters (PALL- RCPL or RC2D) obtained from the Red Cross Blood Bank Facility (Nashville, TN) as described in Meyer et al., 2005 (link). Leukocytes were retrieved from the filters by back-flushing them with an elution medium (sterile PBS containing 5 mM disodium EDTA and 2.5% [w/v] sucrose) and collecting the eluent. The eluent was layered onto lymphosep cell separation media (1.077g/mL) ((MP Biomedicals, Irvine, CA) and centrifuged at 1200g for 30 min. Mononuclear cells were collected and washed with phosphate buffered saline (PBS) pH 7.4 (500g, 10min). Following washing, the cells were layered on bovine calf serum for platelet removal. The cells were then suspended in RPMI-1640 (Mediatech, Inc, Manassas, VA) complete medium which consisted of RPMI-1640 supplemented with 10% heat-inactivated BCS, 2 mM L-glutamine and 50 U penicillin G with 50 μg streptomycin/mL. This preparation constituted PBMCs. Monocyte-depleted PBMCs (10–20% CD16⁺, 10–20 % CD56⁺, 70–80% CD3⁺, 3–5% CD19⁺, 2–20% CD14⁺) were prepared by incubating the cells in glass Petri dishes (150 X 15 mm) at 37 °C and air/CO₂, 19:1 for 1 h. This cell preparation is referred to as MD-PBMCS cells. All cell isolation procedures were carried out under sterile conditions.

Peripheral blood (buffy coat) used in this study was from healthy adult volunteer donors (Red Cross, Portland, OR and Key Biologic LLC, Memphis, TN). Highly purified NK cells were obtained using a rosetting procedure. NK cells were purified by mixing 30–40 mL of buffy coat with 0.6 mL of RosetteSep human NK cell enrichment antibody cocktail (Stem Cell Technologies, Vancouver, BC, Canada). The mixture was mixed and incubated for 20 min at room temperature (25 °C). 7 – 9 mL of the mixture was layered onto 4 mL of Lymphosep cell separation media (1.077 g/mL; MP-Biomedicals, Santa Ana, CA) and centrifuged at 1200g for 30–50 min. The cell layer was removed and washed twice with phosphate buffered saline (PBS, pH 7.2) and stored in complete media (RPMI-1640 supplemented with 10% heat inactivated bovine calf serum (BCS), 2 mM L-glutamine and 50 U penicillin G with 50 mg streptomycin/mL) at 1 million cells/mL.