Deltavision personal

CAR T-cells were incubated with 0.05 µg/mL biotinylated Protein L for 45 min on ice, then washed twice with PBS. Cells were fixed with 4% (w/v) Paraformaldehyde Cytofix Fixation Buffer, for 15 min at room temperature and then washed. Cells were stained with 0.3 µg/mL Streptavidin-APC, 4 µg/mL anti-CD3 UCHT1 Brilliant Blue 515 (Table 1) and 0.2 μg/mL 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, #D1306, Invitrogen). After washing, cells were resuspended in a 50 µL of Vectashield antifade mounting media (#H-1000-10, Vector Laboratories) and added to a well in an 8-well Ibitreat µ-Slide (#80826, Ibidi). Cells were imaged on a DeltaVision Personal (Applied Precision) microscope using the 40 × objective.

Transduced K562 cells (1 × 10⁶) were fixed with an equal volume of 4% (w/v) formaldehyde for 20 min at room temperature (RT). Cells were centrifuged at 2000 rpm for 3 min and resuspended in PBS twice. Cells (5.0 × 10⁵) were allowed to settle onto coverslips coated with poly-d-lysine, before drying and permeabilisation with Triton X-100 0.5% (v/v) in PBS for 10 min at RT. Cells were rinsed three times in PBS and blocked in 3% (w/v) BSA/PBS for 40 min at RT. Cells were rinsed three times in PBS and incubated with mouse anti-HA antibody (1:500, HA.11, Covance) for 90 min at RT. Cells were rinsed three times in PBS and incubated with F(Ab’)2-goat anti-mouse IgG-Alexa 594 (1:500, #A11020, ThermoFisher Scientific) and DAPI (1:10,000, #D1306, Life Technologies) for 40 min at RT. Cells were rinsed three times in PBS and mounted using Prolong Gold Antifade (Life Technologies). Slides were imaged at 60 × using the DeltaVision Personal (Applied Precision) and the DAPI, FITC and A594 filters. Images were analysed after deconvolution using Volocity software.