Cd86 clone ha5.2b7

CD14⁺ monocytes were isolated from PBMCs by positive selection. Isolated monocytes (10,000 cells) were plated in a 96-wells flat-bottom plate. 24 hours later, 10,000 CAR T cells and 10,000 tumor cells (MM1.S) were added in each well and were incubated for 24 hours. After incubation, the wells were washed with phosphate-buffered saline twice and detached from the wells with Trypsin/EDTA solution (Lonza) for 20 minutes. Monocytes were stained with CD3 (clone SK7, BD Biosciences), CD14 (clone HCD14, BioLegend), HLA-DR (clone G46-6, BD Biosciences), CD86 (clone HA5.2B7, Beckman Coulter), and CD80 (clone 16-10A1, BioLegend) (staining with 1 μg per mL of sample, incubation for 30 minutes at 4°C in PBS with 1% BSA, 10% FBS and 0.1% NaN3 sodium azide). Flow cytometry was performed on BD LSR Fortessa.