As originally described,
3 HEK293 cells were grown on poly-L-lysine-coated 25 mm round glass coverslips and co-transfected with cDNA encoding enhanced green fluorescent protein (GFP) (peGFP-C1; Clontech, Burlington, Ontario, Canada) and pcDNA 3.1 (empty vector) or the
SLC4A11 plasmid constructs (WT, E143K and G709E) in a 1:8 molar ratio. cDNA for SLC4A11 variants encoded a shortened version of splicing variant 2 of human SLC4A11 (891 amino acid, NCBI reference: NG_017072.1) with an N-terminal HA tag, as previously described previously.
20 One hour post transfection, cells were treated with the final concentration of drug in 0.2% DMSO (
Table), and control cells received DMSO to a final concentration of 0.2% DMSO (vol/vol). Forty-eight hours later, coverslips were mounted in a 35-mm diameter Attofluor Cell Chamber (Molecular Probes, Ottawa, ON, Canada) and washed with PBS. During experiments, the chamber was perfused with isotonic MOPS buffered saline solution (MBSS) (90 mM NaCl, 5.4 mM KCl, 0.4 mM MgCl
2, 0.4 mM MgSO
4, 3.3 mM NaHCO
3, 2 mM CaCl
2, 5.5 mM glucose, 100 mM D-mannitol, and 10 mM HEPES, pH 7.4, 300 mOsm/kg), and then with hypotonic (200 mOsm/kg) MBSS buffer, pH 7.4 (same composition as previous but lacking D-mannitol). Solution osmolarity was measured using an osmometer (Advance Instruments, Inc., Norwood, MA, USA). The chamber was mounted on the stage of a Wave FX spinning disc confocal microscope (Quorum Technologies, Guelph, ON, Canada), with a Yokogawa CSU10 scanning head (Tokyo, Japan). The microscope has a motorized XY stage with Piezo Focus Drive (MS-4000 XYZ Automated Stage; ASI, Eugene, OR, USA) and a live-cell environment chamber (Chamlide, Seoul, Korea), set to 24°C during the experiment. Acquisition was performed with a Hamamatsu C9100–13 Digital Camera (EM-CCD; Chamlide) and a ×20 objective during excitation with a laser (Spectral Applied Research, Richmond Hill, ON, Canada) at 491 nm. GFP fluorescence, collected through a dichroic cube (Quorum Technologies) at wavelengths 520 to 540 nm, was acquired at 1 point s
−1 for 4 minutes. Quantitative image analysis was performed by selecting a region of interest for each HEK293 cell with Volocity 6.0 software (PerkinElmer, Waltham, MA, USA). Following the switch to hypotonic MBSS buffer, the rate of fluorescence change was determined from the initial 15 seconds of linear fluorescence change.