Liquid scintillation analyser

ES cells were plated in 12-well plates (2.4-cm diameter/well) and the ⁸⁶Rb⁺-uptake assay was performed on cells that were 80% confluent. HEK-293 cells were plated at a confluence of 50–60% in 12-well plates (2.4-cm diameter per/well) and transfected with wild-type or various mutant forms of full-length FLAG-tagged human KCCs. Each well of HEK-293 cells was transfected with 2.5 μl of 1 mg/ml polyethylenimine and 1 μg of plasmid DNA. The ⁸⁶Rb⁺-uptake assay was performed on the cells at 36 h post-transfection. In both cases, culture medium was removed from the wells and replaced with either isotonic or hypotonic medium for 15 min at 37°C. Cell medium was removed by means of aspiration with a vacuum pump and replaced with stimulating medium plus inhibitors including 1 mM ouabain and 0.1 mM bumetanide, to prevent ⁸⁶Rb⁺ uptake via NKCC1, for a further 15 min. After this period, the medium was removed and replaced with isotonic medium plus inhibitors containing 2 μCi/ml ⁸⁶Rb⁺ for 10 min at 37°C. After this incubation period, cells were rapidly washed three times with the respective ice-cold non-radioactive medium. The cells were lysed in 300 μl of ice-cold lysis buffer and ⁸⁶Rb⁺-uptake tracer activity was quantified on a PerkinElmer liquid scintillation analyser.

IUR [40 (link)] were performed in triplicate with 2 × 10⁵ cells per tube. Briefly, 2 × 10⁵ cells were incubated for 2 h at 37 °C in the presence and absence of a 50% ¹⁴C-labeled 2 μM AZA. For IUR with inhibitors, 100 μM verapamil (Royal Adelaide Hospital Pharmacy, Adelaide, SA, Australia), 200 μM procainamide (Sigma-Aldrich, St. Louis, MO, USA), 10 μM corticosterone (Sigma-Aldrich, St. Louis, MO, USA), 20 μM NBMPR (Sigma-Aldrich, St. Louis, MO, USA), 20 μM cyclosporin A (Sigma-Aldrich, St. Louis, MO, USA), 10 μM chloroquine (Sigma-Aldrich, St. Louis, MO, USA), 150 μM amantadine (Sigma-Aldrich, St. Louis, MO, USA), 20 and 200 μM cimetidine (Sigma-Aldrich, St. Louis, MO, USA), and 0.1 and 10 μM pyrimethamine (Sigma-Aldrich, St. Louis, MO, USA) were added. After incubation the cellular and aqueous phases were separated, and incorporation determined using a Perkin Elmer Liquid Scintillation Analyser following the addition of Microscint 20 scintillation fluid (Perkin Elmer, Waltham, MA, USA) before counts per minute of β radiation in the supernatant and cell pellet fractions was used to convert to ng of AZA in 2 × 10⁵ cells. All assays were performed in triplicate and repeated if the assay demonstrated non-concordance.

Flotillin 2 was cloned into AgeI and EcoRI sites of pU39 with a C-terminal transmembrane domain of monoamine oxidase (MOA), which is sufficient for targeting to the outer mitochondrial membrane [34 (link), 47 (link)]. HeLa cells were transfected with flotillin 2-MOA (‘flot2-mito’) and flotillin-1-GFP. Mitochondrial targeting was determined by confocal microscopy using 20 nM MitoTracker Deep Red (Molecular probes). To elucidate sphingosine recruitment on mitochondria, 10⁷ Hela cells were transiently transfected, the cells were harvested and chilled on ice, and incubated with 1 μCi/ml ³H-Sph for 5 min. After washes with cold PBS, mitochondria were purified using Mitochondria isolation kit (Thermo Scientific) and ³H-Sph was quantified with liquid scintillation analyser (Perkin Elmer).