A1 confocal module

Immunofluorescence was used to visualize differentiated cells. Cells grown on poly-d-lysine were fixed with 4% paraformaldehyde in PBS. After washing in PBS, cells were permeabilized in 0.1% Triton X-100/PBS/0.1% FBS for 30 min at 4 °C and again washed twice with PBS/0.1% FBS. Then samples were incubated with 0.1% Triton X-100, PBS, and 1% FBS in PBS for 2 h. To identify nuclei, cells were incubated with 1 µg/mL Hoechst 33342 (Thermo Fisher Scientific). To assess cell differentiation, cells were incubated with anti-neural cell adhesion molecule monoclonal antibodies 1:200 in PBS containing 1% FBS and Triton X-100 (NCAM; 56C04; Thermo Fisher Scientific). Then, samples were treated for 1 h with rabbit anti-mouse IgG H + L conjugated with Alexa Fluor^® 546 (1:1000; Thermo Fisher Scientific), washed PBS containing 1% FBS and Triton X-100, and then stained with antibodies to β-III tubulin conjugated with Alexa Fluor 647 (BioLegend; 1:100). The images were obtained using an Eclipse Ti-E microscope with an A1 confocal module (Nikon Corporation, Tokyo, Japan) and a CFI Plan Apo VC 20×/0.75 objective. Data were visualized using Nikon proprietary software (NIS-Elements).

Cells were placed on poly-D-lysin coated glasses and fixed with 4% paraformaldehyde in PBS, washed with PBS, permeabilized in 0.1% Triton X-100/PBS/0.1% FBS for 30 min at 4°C, and blocked with PBS/1% FBS/0.1% Tween-20 (PFT). To identify CAR19 distribution cells were stained with CD19 CAR Detection Reagent (1:600 in PFT) for 12 h, then samples were washed with PFT and treated for 1 h with anti-biotin-PE (Thermo Fisher Scientific, 1:1000). To assess PD1 expression, samples were treated for 12 h with anti-PD1 antibodies, conjugated with FITC (1:500 in PFT). For nuclei counterstaining 10 µM Hoechst 33,342 was used. Images were captured on an Eclipse Ti-E microscope with the A1 confocal module (Nikon Corporation) and Apo TIRF Plan Fluor 63 × 1.49.