Flow cytometry cell staining was performed at 4°C in PBS supplemented with 2% FBS, unless otherwise indicated. The expression of MSLN on cancer cells was detected by flow cytometry, using a human MSLN PE-conjugated antibody (R&D, FAB32652P, USA).
The expression of CAR on CAR-T cells was detected using biotinylated human MSLN (Acro, Beijing, China), followed by staining with allophycocyanin (APC) streptavidin (BioLegend, CA, USA).
The immunophenotypes of T cells were tested using flow cytometry. The antibodies used for analysis include: APC-Cy 7 mouse anti-human CD3 (BD, NJ, USA), APC mouse anti-human CD4 (BD, NJ, USA), phycoerythrin (PE) mouse anti-human CD8 (BD, Franklin Lakes, NJ, USA), Brilliant Violet 421 anti-human CD197 (BioLegend, CA, USA), fluorescein isothiocyanate (FITC) anti-human CD45RA (BioLegend, San Diego, CA, US), PE-Cy7 anti-human CD45RO (BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD197 (CCR7; BioLegend, San Diego, CA, USA), Brilliant Violet 510 anti-human CD62L (BioLegend, San Diego, CA, USA), Brilliant Violet 605 anti-human CD95 (Fas; BioLegend, San Diego, CA, USA), PE anti-human CD223 (LAG-3; BioLegend, San Diego, CA, USA), Brilliant Violet 421 anti-human CD366 (Tim-3; BioLegend, San Diego, CA, USA), and APC anti-human CD279 (PD-1; BioLegend, San Diego, CA, USA). Data were analyzed using NovaExpr software (ACEA, Ashland, OR, USA).
Zhang Q., Liu G., Liu J., Yang M., Fu J., Liu G., Li D., Gu Z., Zhang L., Pan Y., Cui X., Wang L., Zhang L, & Tian X. (2021). The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice. Molecular Therapy Oncolytics, 20, 556-568.
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