Sepmate 50 ml tubes

For all critically ill and non-severe patients with viral and bacterial induced sepsis, blood was collected into 10 mL EDTA tubes (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and loaded using SepMate 50 mL tubes (Stemcell Technologies, Saint-Egreve, France). Gradients were centrifuged at 1200× g for 10 min with the brake on at room temperature. The cell interface was carefully removed by pipetting and washed with PBS-EDTA by centrifugation at 400× g for 7 min. PBMC pellets were suspended in an ammonium chloride solution (Stemcell Technologies, France) and incubated for 10 min at room temperature on a mixing platform to lyse contaminating red blood cells. The lysed pellet was resuspended in 10 mL of PBS (a small sample was taken for counting) and underwent spin suspension at 400× g for 7 min with the brake on. Isolated PBMCs were finally washed with PBS-EDTA and then resuspended for downstream analyses. The blood was processed within 4 h of collection for all samples. All samples obtained on the day were processed side-by-side to avoid variation from processing. They were analyzed fresh on the BD Rhapsody Single-Cell Analysis System platform to avoid clumpy cells and achieve a higher viability % of cells.

Whole blood from healthy females (age 18–55) was purchased from Zen Bio (Durham, NC). Monocytes were enriched using RosetteSep Human Monocyte Enrichment Cocktail in combination with SepMate 50 mL tubes according to the manufacturer’s instructions (STEMCELL Technologies, Seattle, WA). Human HGSOC cell lines OV90 and OVCAR3 were purchased from ATCC (Manassas, Virginia), OVCA432 and OVCAR5 were obtained from Dr. R. Bast (MD Anderson Cancer Center; Houston, TX, USA), and OVCAR4 were obtained from the NCI Tumor Repository (Frederick, MD). All ovarian cancer cell lines were maintained in ovarian cancer media (1:1 (v/v) ratio of MCDB105: Medium199 (Corning, Corning, NY) supplemented with 1% penicillin/streptomycin) with 15% heat-inactivated fetal bovine serum prior to co-culture.