Anti cd86 percp cy5

CAR Mesothelin surface expression on human T-cells was assessed using primary antibody goat anti-human IgG F (ab’)2 Biotin (BioRad, Hercules, CA) along with secondary conjugate Streptavidin APC (BioRad). The following antibodies were also used in FACS buffer in this study: anti-PD1-PE (Biolegends 329906), anti-CCR7-Alexa-fluor 647 (BD 560921), anti-CD8-FITC (BC A07756), anti-CD8-BV421 (BD 562428), anti-HLA-A2-FITC (BD 551,285), anti-CD86-PerCp-Cy5.5 (BD 561129), anti-CLDN6-Dylight 650 (IMAB027), anti-idiotype-IMAB027- Alexa-fluor 647, anti-TIM3-APC (Biolegends 345011) and 7AAD (BC A07704). Mesothelin expression was detected using the PE conjugated anti mesothelin antibody (R&D system FAB32652P). Acquisition and analysis of all samples were performed on a BD FACS CantoI/II (BD Biosciences, San Jose, California) and FlowJo software (v7.6.1).

Recombinant human (rh) cytokines, including IL-1α, IL-2, IL-4, GM-CSF, IFN-γ, and TNF-α, and anti-CD3 and anti-CD28 antibodies were purchased from Beijing T&L Biotechnology. Fluorophore-labeled primary antibodies, including anti-CD3-FITC, anti-CD4-phycoerythrin/PE, anti-CD56-allophycocyanin/APC, anti-CD80-PE, anti-CD83-APC, and anti-CD86-PerCP-Cy5.5, were acquired from BD Biosciences; anti-CD44 and anti-EpCAM antibodies were obtained from Abcam. Ficoll-Paque PLUS medium was obtained from GE Healthcare Life Sciences; Lonza X-VIVO™ 15 medium from Fisher Scientific; CCK-8 reagent from Dojindo Molecular Technologies; and TRIzol reagent from Molecular Research Center.

Peripheral blood mononuclear cells (PBMC) were obtained from K2EDTA blood samples. Tubes were centrifuged and plasma was collected, aliquoted and stored at −20 °C. Cells were diluted with phosphate-buffered saline (PBS), PBMC were isolated by Ficoll density gradient and directly used for flow cytometry (FC) analysis and monocyte isolation.
Monocyte phenotype was analyzed by FC (FACS Canto II, BD Bioscience, San Jose, CA, USA). PBMC were stained with anti-CD3-V450, anti-CD19-V450, anti-CD56-V450, anti-CD14-FITC (BD Bioscience, San Jose, CA, USA), anti-CD33-PE-Cy7 (eBioscience, San Diego, CA, USA), anti-HLA-DR-APC, anti-CD16-V500, anti-CCR2-PE, anti-CCR5-APC-Cy7 and anti-CD86-PerCP-Cy5.5 (BD Bioscience, San Jose, CA, USA). Classical monocytes were defined as CD14+CD16−, intermediate monocytes as CD14+CD16+ and non-classical monocytes as CD14−CD16+. FC data were analyzed in FlowJo V10.