Pe conjugated anti human cd14 antibody

Cell components in peritoneal washes were re-suspended in RPMI-1640 supplemented with 10% FBS. Then, the total number of viable cells was counted, and the cells were infected with TelomeScan at 1 MOI for 24 hours at 37ºC to visualize cancer cells as GFP-positive cells.^24–26 The cells were further stained with Alexa Fluor 647-conjugated anti-human CD45 antibody (BioLegend, 304056, RRID: AB_2564155), phycoerythrin (PE)-conjugated anti-human CD14 antibody (BioLegend, 301805, RRID:AB_31418), and PE-conjugated anti-human CD163 antibody (BioLegend, # 333606, RRID:AB_1134002) as markers of leukocytes, macrophages, and TAMs, respectively. Nuclei were stained with 4ʹ, 6-diamidino-2-phenylindole (DAPI). Immunofluorescent staining was observed under an inverted fluorescence microscope (IX71; Olympus).

About 25 mL of the fasting anti-coagulated blood sample was diluted with an equal volume of lymphocyte separation solution (Nakalai tesque, Japan). The samples were centrifuged at 400 g, in a swing out rotor for 30 min at 22°C, and the peripheral blood mononuclear cell (PBMC) layers were harvested using a pipette. PBMCs were repeatedly washed using Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific Inc., Waltham, MA, USA) with 1% penicillin/streptomycin (P/S) containing 10% fetal bovine serum (FBS). PBMCs were cultured on collagen-coated multi-well plates in DMEM with 1% P/S containing 10% FBS for 2 h in an incubator. PBMC was washed with phosphate-buffered saline (PBS) to remove lymphocytes.^{8 ,9 (link)} Purity of the monocytes was verified by a FACSCelesta (Becton, Dickinson and Company, NJ, USA), using phycoerythrin (PE)-conjugated anti-human CD14 Antibody (BioLegend, CA, USA) (Supplemental Figure 1). For serum starvation, the media was replaced with DMEM containing 0.25% bovine serum albumin (BSA) for 2 h. Cultured monocytes were stimulated by 50 nM insulin for 5 min or 10 ng/mL LPS for 4 h on 12-well plates.