Cd45ra pe

Changes in T cell phenotype by flow cytometry were performed to analyze activation and memory surface markers before and after T cell encapsulation using BD Accuri C6 Plus equipment (BD Bioscience, Franklin Lakes, NY, USA). Three and four multicolor flow cytometry panels were employed to study the T cell phenotype changes. Zombie NIR fixable cell viability dye (BioLegend, San Diego, CA, USA) was used to discriminate the dead cell population in both panels. To identify the activated T cell populations, APC-CD8, PE-CD69, and Kiravia blue HLA-DR labeled antibodies (BioLegend, San Diego, CA, USA) were employed. Similar to the activation panel, the memory panel also included APC-CD8 along with FITC-CD45RA labeled antibody (BioLegend, San Diego, CA, USA). In both panels, labeled antibodies were employed according to the manufacturer's instructions at 5 μl antibodies per 1 M cells in a 100 μL sample. Moreover, the CAR content of the T cell population was identified using 400 ng/10⁶ T cells of 1A7 anti-14G2a idiotype antibody (Absolute Antibody, Boston, MA, USA) conjugated with APC Conjugation Kit – Lightning-Link (Abcam, Waltham, MA, USA). The 1A7 anti-14G2a idiotype antibody was employed along with FITC-CD8 labeled antibody to form 3 panels with PE-CD45RA, PE-CD45RO, and PE-HLADR (BioLegend, San Diego, CA, USA) for the CAR-cell phenotype characterization.