Pd l1 pe 10f 9g2

To detect the PD-L1 expression on GL161 cells, cells were detached using Accutase (Thermo Fisher Scientific, USA) and then stained with PDL1-SPIO nanoparticles, SPIO nanoparticles, PDL1 antibodies, or isotype control antibodies, followed by PE-conjugated anti-rat IgG2b antibodies (MRG2b-85, BioLegend, USA). To detect PD-L1 expression on brain-infiltrating leukocytes, cells were stained with specific antibodies following standard protocols and analyzed on a CytoFLEX flow cytometer (Beckman Coulter, USA). The following antibodies conjugated with fluorochrome were used: Ly6G-PB (RB6-8C5), Ly6C-FITC (HK1.4), CD45-APC-Cy7, (30-F11), CD11b-PB (M1/70), and PD-L1-PE (10F.9G2) (BioLegend, USA). Data were analyzed using FlowJo software.

Cell suspensions were Fc blocked using anti-CD16/32 mAbs (clone 93; BioLegend) and normal murine serum 1%. Stainings were performed with the following mAbs: hCD34-BV421 or Alexa Fluor 647 (561; BioLegend), CD3e-BUV395 (145-2C11; BioLegend) or CD8-BUV395 (53–6.7; BioLegend), PD-1 Pe/Cy7 (J43; BioLegend), CD19-APC-fire750 (6D5; BioLegend), or PD-L1-PE (10F.9G2; BioLegend). Intracellular stainings were performed on CAR or control T cells cultured in the presence or absence of target cells for 20 h using the Cytofix/Cytoperm kit (BD Biosciences), Allophycocyanin-conjugated anti-Granzyme B mAb (GB11; BioLegend) or Alexa Fluor 647–conjugated anti-IFN-γ mAb (clone XMG1.2; eBioscience). IFN-γ expression was assayed in the presence of 1 µg/ml Brefeldin A (BD Biosciences). Analyses were performed with a FACSCanto II cytometer (BD Biosciences) or an LSR/Fortessa (BD Biosciences) and analyzed with FlowJo software version 10.1 (Tree Star). FRET loss was defined as a derived parameter using the ratio of CFP to FRET fluorescence. FRET loss was measured on cells fixed with 2% paraformaldehyde solution (Sigma) immediately after isolation.