Facs symphony a5 instrument

PBMCs were thawed as described above. For measurement of chemokine receptor expression, cells were stained immediately after thawing with fluorophore-tagged antibodies directed against chemokine receptors for 15 minutes at 37 degrees, then fluorophore-tagged antibodies directed against other cell surface molecules were added and cells were further stained for 15 minutes at 4 degrees. After antibody staining, non-viable cells were labelled with Live/Dead Fixable dye (ThermoFisher) and acquired using a FACS Symphony A5 instrument (BD). For measurement of cytokine production, once PBMCs were thawed they were allowed to rest for 2 hours to recover from cryopreservation before 4 hours of stimulation with 1µg/mL ionomycin, 10ng/mL PMA, 1µg/mL brefeldin A and 2µM monensin. Non-viable cells were labelled with Live/Dead Fixable dye and incubated with a panel of fluorophore-tagged antibodies directed against surface markers. Cells were then fixed and permeabilized using the CytoFix/CytoPerm kit (BD) according to manufacturer’s instructions and stained with a panel of fluorophore-tagged antibodies directed against intracellular cytokines before being acquired using a FACS Symphony A5 instrument.

Tumor and non-tumorous liver tissue from HCC patients was manually cut into small tissue fragments of 1-2 mm³. After processing, tissue fragments were placed in ultra-low adherence Nunclon™ Sphera™ 96-Well U-Shaped-Bottom Microplates in 100 μL of complete medium (RPMI-1640, 10% FBS, 1X GlutaMax, 1X Penicillin/Streptomycin, 1X Hepes, 1X non-essential amino acids, 1X Sodium Pyruvate, 0.01% β-mercaptoethanol). CD14⁺ cells were isolated from peripheral blood from the same patients. CD14⁺ cells were stained using the CellTrace Far Red (CTFR) Cell Proliferation kit (Biolegend) according to the manufacturer's protocol and 50,000 cells CTFR⁺ CD14⁺ cells were added to the wells containing tissue fragments. CTFR⁺ CD14⁺ cells were cultured alone as controls. After 3-7 days of co-culture cells and tissues were recovered, disaggregated mechanically, and filtered through 40 μm cell strainers (BD). Cell suspensions were stained with Zombie UV viability dye (Biolegend), anti-THY1 PE (1/50, Thermo Fisher), anti-CD45 PerCP-Cy5.5 (1/100, Miltenyi), anti-CD11b APC-Vio770 (1/100, Miltenyi) and anti-CD14 V500 (1/50, BD). Stained cells were analyzed using a FACS Symphony A5 instrument (BD).