Ti e microscope

TIRF experiments were performed as described previously^{53 (link)}. In brief, 3T3-L1 adipocytes at day 7 were trypsinized, electroporated with the construct of interest and seeded onto Matrigel coated 35 mm µ-dishes (Ibidi). After 24 h, cells were incubated with basal medium (DMEM without FBS) for 2 h. Following this media was replaced with KRP + buffer (KRP, 10 mM glucose and essential amino acids) and dishes were placed onto the stage of a Nikon TiE microscope equipped with an OKOlab microscope enclosure maintained at 37 °C. TIRF was achieved using a Nikon hTIRF module. TagRFP-T was stimulated with a 568 nm laser angled at 71 °C and emission was captured on an Andor 888 emCCD camera after passing through a 610/50 nm filter. Buffer switching was performed using a custom fluidic setup. Cells were treated with insulin, 100 μM BCNU and 1 μM AF, 100 μM H2O2, 50 mU/ml glucose oxidase (GOD), 10 μM diphenylene iodinium (DPI), where added as indicated. Image data were analysed using Fiji^{89 (link)}.

HEK293 cells were imaged 2 days after transfection with fluorescence-tagged HO variant or in co-transfection with HA-SPP or HA-SPP mut at 37°C on a Nikon Ti-E microscope equipped with an incubation chamber (Okolab) using a 60 x oil immersion objective (NA 1.4, Nikon). After exposure to hypoxia (1% O₂) for 48 h samples only transfected with fluorescence-tagged HO variants were imaged again. A focussed 488 nm laser was used for GFP excitation. GFP emission was measured between 500–550 nm. CFP and YFP were excited with 457 nm and 514 nm, the corresponding emissions were measured between 464-499 nm and 525–555 nm.