Facs cantoi 2

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).

CD4 and CD8 T cells were depleted from PBMC by positive selection using EasySep positive selection cocktail containing monoclonal antibodies coupled to Magnetic Nanoparticles (Stemcell Technologies, Vancouver, BC, Canada). After the cell separation total CD4 and CD8 T cell subsets were assessed by flow cytometry. Briefly, 1,000,000 cells were stained in 5 mL Falcon round bottom tubes with human anti-CD4 FITC and CD8 PE (all BD Biosciences, San Jose, CA, USA) and fixed in 2% formaldehyde. A minimum of 200,000 events were acquired using BD FACSCanto III (BD) and analyzed with FACSDivaTM, version 4 software.

Surface expression of CD44 and CD133 markers was detected using flow cytometry (FC). The cells were harvested and stained with fluorochrome-conjugated antibodies: anti-CD44-FITC (BD Biosciences, San Jose, CA, USA) and anti-CD133-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Protein expression was measured for a minimum of 10,000 events using BD FACS Canto III (BD Biosciences). Detection of a side population was done with Hoechst 33,342 dye (Sigma-Aldrich) by BD FACS Aria III (BD Biosciences).