Opera phenix platform

Cells were grown on glass coverslips and cultured/treated as described above. ARPE19 mito‐QC cells were fixed in 3.7% PFA at pH 7.0 in 0.2 M HEPES, counterstained with Hoechst 33342 and VECTASHIELD Antifade Mounting Medium H‐1000 was used to mount coverslips on slides. Images were acquired using an ANDOR Spinning Disc Microscope equipped with a Zyla camera (Plan Apochromat ×40 objective, NA 1.15). For high‐content temporal analysis of LD biogenesis, ARPE19 cells were seeded on Ibidi black wall m plates (24 well format) with culture and treatment conditions as described above. Samples were labelled with BODIPY and MitoTracker and fixed as described above, followed by imaging with a PerkinElmer Opera Phenix Platform. For analysis of LDs in ULK1 KO cells, images were acquired using a wide‐field Nikon Eclipse Ti wide‐field microscope using a Nikon Plan Apo ×60 oil immersion objective.

10,000 cells were seeded per well on black PhenoPlate^™ 96-well microplates (Perkin Elmer Cat# 6055302) and left to settle for 2 days, before being exposed to 10 Gy of gamma irradiation or left untreated (controls). The Click-iT^™ EdU Cell Proliferation Kit for Imaging (Alexa Fluor^™ 555 dye) was used to measure cycling cells according to manufacturer’s protocols. Cells were then stained for RAD51 and γH2AX foci and imaged on the Perkin-Elmer OPERA PHENIX platform as previously described (17 (link)).