Fig 1: Assessment of anti-canine CD20 CAR expression and transduction efficiency(A) Reagents used to label UTD and CAR cells. UTD and CAR cells were treated with 10 µL of dog gamma-globulins in ~ 100 µL for 10 min at 20°C–25°C, followed by incubation with 0.5 µL (0.5µg) of Biotinylated Rabbit anti-mouse (RaM) IgG H+L for 30 min at 20°C–25°C (first step). At the end of incubation, the unbound immunoglobulins were removed by adding 4 mL FACS buffer, followed by 5-min centrifugation at 500 ×g and complete removal of the supernatant by decanting the supernatant and gently touching the rim of the tubes to tissue paper, for a total of two washes. T cells were incubated with anti-dog CD3, CD5, CD4 and CD8 mAb and Streptavidin for 20–30 min at 2°C–8°C (second step). At the end of incubation, cells were washed once as above and resuspended in 300 µL FACS buffer with 2 µL 7AAD prior to data acquisition by flow cytometer.(B) Representative plots showing CAR transduction efficiency relative to UTD cells.