Fluoview fv3000 confocal microscope system

Cells were seeded into ibidi 8-well µ-Slide (Ibidi, Germany) in media. After 24 h, media was removed and cells received their treatment. After treatment cells were washed and fixed in 4% paraformaldehyde. Cells were then blocked and permeabilised in blocking buffer (1X PBS + 5% BSA + 0.3% Triton X-100). Cells were incubated in primary phospho-histone γ-H2AX (Ser139) rabbit mAb (1:200 dilution in blocking buffer; 9718, Cell Signaling Technology) overnight at 4 °C in the dark. Cells were then washed before being incubated in the secondary antibody Anti-rabbit IgG (H + L), F(ab')2 Fragment - Alexa Fluor® 488 Conjugate (4408, Cell Signaling Technology); 1:1000 dilution in blocking buffer. After washing cells were counterstained with DAPI (0.5 µg/ml) for 5 minutes. Images were acquired using an Olympus Fluoview FV3000 confocal microscope system using a ×60 objective. All experiments were performed in triplicate with at least three independent experiments.

For the in vivo experiments, FRAP of EGFP-Bex1 in ARPE19 cells was performed on Fluoview FV3000 confocal microscope system (Olympus). Using a 60× oil immersion objective, a whole EGFP-Bex1 granule was bleached using a laser intensity of 20% at 488 nm. Recovery was recorded for every second for a total of 300 s after bleaching. Analysis of the recovery curves was carried out with cellSens software (Olympus).
For the in vitro experiments, FRAP was carried out with samples in glass bottom 8-well chamber slides using a Fluoview FV3000 confocal microscope equipped with 60× oil immersion objectives, as above. Condensates were bleached using a laser intensity of 30% at 561 nm. Recovery was recorded for every second for a total of 180 s after bleaching. Analysis of the recovery curves was carried out with cellSens software (Olympus).