Rpmi 1640 with glutamine

Peripheral blood mononuclear cells (PBMCs) were harvested in BD Cell Preparation Tubes (Becton Dickinson and Company) at enrollment, steady state (defined as 30 days after first injection for DMPA with a mean serum MPA concentration of 1303.5 pg/mL or 90 days post-injection for Net-En with mean serum NET concentration of 1565.5 pg/mL) and nadir (period immediately prior to next clinical dosing at day 180 visit and lower mean serum concentration of 279 and 856 pg/mL for MPA and NET, respectively). PBMCs were cryopreserved in 90% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO; Sigma Aldrich) and stored in liquid nitrogen until analysis and then quick thawed by gentle agitation in a 37°C water bath followed by drop-wise addition of pre-warmed culture medium (CM) containing 1-part FBS and 9-part RPMI 1640 with glutamine (Sigma Aldrich). Cells were washed by centrifugation at 500 g for 8 minutes and re-suspended to 1 × 10⁶ cells/mL in CM and rested overnight at 37°C in 5% CO₂. Cell viability was determined by staining cells using 0.4% trypan blue and counting on a hemocytometer under microscope.²⁰ Cell viability after recovery was >95% for all specimens before stimulation and >80% after stimulation procedures.

Human embryonic kidney (HEK) 293 cells were cultured at 37°C in DMEM supplemented with 10% FBS (Gibco), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (Sigma-Aldrich). HEK293 cells were seeded in 24 wells plates (Luciferase assay) or 12 wells plates (immunoblotting) and cultured for 16 to 24 hours before transfection using PolyJet (SignaGen) and equal amounts of DNA.
Peripheral blood mononuclear cells (PBMCs) were isolated by gradient Ficoll centrifugation (Sigma-Aldrich). For the analysis of SYK and pSYK by immunoblotting PBMCs were cultured overnight in RPMI1640 supplemented with 20% FBS.
For ex vivo T cell phenotyping, PBMCs were cultured in RPMI-1640 with glutamine (Sigma-Aldrich) supplemented with 5% human serum (NHS Blood Center Oxford), 1% sodium-pyruvate, 1% non-essential amino acids (Gibco), 1% penicillin/streptomycin (Gibco), afterwards referred to as complete medium.