Du 897 emccd camera

Cells plated on 96-well glass-bottom plates were incubated at 37°C and 5% CO₂ by an Okolab microscope stage incubator with 96-well insert during all imaging experiments. Confocal microscopy was performed on a spinning disk (Yokogawa CSU-X1) confocal microscope with an Andor DU-897 EMCCD camera on a Nikon Eclipse Ti body using a 100x oil immersion Apo TIRF objective (NA 1.49). The following wavelength lasers were used to image the respective constructs: constructs with mGFP (488 nm), mCherry (561 nm), miRFP (640 nm). Fixed samples in the 53BP1 counting assay also used the 405 nm laser to detect nuclei stained with Hoechst (Thermo Fisher Scientific, H3570) or DAPI (Vectashield, H-2000-10).

Coverslips were pretreated with poly D-lysine, and then cells were plated onto coverslips at sub-confluence. Cells were fixed in 4% methanol-free formaldehyde for ten minutes and permeabilized with 0.5% Triton for ten minutes. Proximity ligation assays were performed with the Duolink PLA mouse/rabbit kit (Sigma) according to the manufacturer’s instructions. Nuclei were stained using Duolink in situ mounting medium with DAPI (Sigma DUO82040). Primary antibodies used were mouse anti-MYC (Santa Cruz c33) and rabbit anti-BAF155 (Abcam ab72503). Confocal images were acquired using an Andor DU-897 EMCCD camera mounted on a Nikon Spinning Disk Microscope. Fluorescent puncta were quantified by using FIJI (ImageJ) to calculate the number of maxima within each nucleus.

TCR-activated T cells were labelled with 1μm Oregon green BAPTA-488 (ThermoFisher; O6807) prior to stimulation with planar lipid bilayers as described above. Cells were imaged using an inverted TiE microscope, equipped with a 1.4× 40X oil objective, Lambda LS lightsource and DU-897 EMCCD camera controlled by and analyzed with, NiS Elements software (Nikon Instruments).

Spheroids were fixed in 4% formaldehyde in PBS for 30 min at 4˚C then transferred into 2.0 mL tubes, rinsed three times in 0.1% TBST, permeabilized with 0.5% TBST for 30 minutes on a rotator at RT, rinsed with 0.1% TBST, and incubated in blocking buffer for 1 hour on a rotator at RT. Spheroids were then incubated with mouse anti-β-catenin antibody (1:500, Vanderbilt Protein and Antibody Resource) or rabbit anti-E-cadherin antibody (1:200, Cell Signaling Technology) in blocking buffer overnight on a rotator at 4˚C. After incubation, spheroids were washed three times for 30 minutes per wash in 0.1% TBST and incubated 2–3 hours on a rotator at RT with Cy3-conjugated anti-mouse secondary antibody (1:500, Jackson Immune) or Cy3-conjugated anti-rabbit secondary antibody (1:500, Abcam) diluted in blocking buffer. Spheroids were rinsed in 0.1% TBST then incubated with Alexa Fluor 568 Phalloidin (1:1000; ThermoFisher Scientific) and Hoescht (1:1000) in 0.1% TBST for 20 minutes on a rotator at RT. Spheroids were rinsed 1X in 0.1% TBST. Spheroids were put into 1X PBS (Vanderbilt Molecular Biology Resource) then mounted on microscope slides with ProLong Gold Antifade Reagent (Invitrogen). Images were acquired using a Nikon Spinning Disk microscope with Andor DU-897 EMCCD camera with 561 nm and 405 nm lasers. Images were processed using ImageJ.