Hek293h cells

The recombinant AAV vectors were produced by calcium phosphate transfection of human embryonic kidney cells (HEK 293-H cells, Thermo Fisher Scientific Waltham, MA, USA) using a three-plasmid based production protocol (AAV helper free system; Agilent Technologies, Santa Clara, CA, USA) and purified as described [99 (link)] Vector yield was analyzed by ddPCR, and viral titers were determined by ELISA (Progen, Heidelberg, Germany) according to the manufacturer’s manual.

HEK293H cells (ThermoFisher Scientific, Waltham, MA, USA, 11631-017) were maintained as a monolayer in DMEM (Gibco, 21063-029) containing 1× MEM non-essential amino acids (Gibco, 11140-035), 1× penicillin-streptomycin (Gibco, 15140-122) and 10% FBS (Gibco, 26140) in a 37 °C incubator with 5% CO₂. HEK293H cells stably expressing the chimeric G protein subunit Ga16gust44 [33 ] were used for the expression of hTAS2R8. The reverse transfection method was adapted from the Sabatini lab description [1 (link)]. In short, we prepared a transfection mix from the Effectene kit with transfection reagents (Qiagen, 301425) by mixing 75 µL EC buffer with 8 µL enhancer, pre-incubating for 5 min at room temperature and then adding 12.5 µL Effectene lipids. The transfection mix was vortexed for 10 s and carefully applied, dropwise, onto the glass surface submerging the entire 15 × 18 spot array. The slides were incubated for 20 min at room temperature. The transfection mix was removed by tilting the array and carefully pipetting off the transfection mix. Once the slides were air-dried, a flexiPERM chamber (Greiner, 90032039) that was cut to the appropriate size was mounted over the array (Figure S1A). The 0.9-cm² chamber was filled from the edge with HEK293H cells (2.2 × 10⁵ cells/cm²) and incubated at 37 °C with 5% CO₂ for at least 27 h before use.

HEK293-H cells (Thermo Fisher Scientific) were maintained in suspension at 37 °C with 5% CO2 in CD293 medium supplemented with 4 mM GlutaMAX (Invitrogen). The HEK293 cell line is a permanent line established from primary embryonic human kidney and transformed with sheared human adenovirus type 5 DNA. For imaging and electrophysiology, cells were plated onto poly-L-lysine–coated coverslips 1 day before transfection and grown in a medium containing 44% Dulbecco’s modified Eagle’s medium (Corning), 44% Ham’s F12 (Corning), 10% fetal bovine serum (HyClone), 2 mM glutamine, 50 U/ml penicillin and 50 μg/ml streptomycin.

HEK293H cells (Thermo Fisher Scientific, Waltham, MA, United States; Catalog ID 11631017) were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; PAN-Biotech, Aidenbach, Germany), 2 mM L-glutamine (Merck Millipore, Darmstadt, Germany), 100 units/ml penicillin, and 100 μg/ml streptomycin (PAN-Biotech) under standard conditions of 5% (v/v) CO₂ and 37°C. Cells were passaged every 3–4 days at around 90% confluency. For transfection, cells were seeded in 100 mm petri dishes with 1.5 × 10⁶ cells per dish and transfected after 24 h with calcium-phosphate precipitation. For this purpose, 22 μg DNA in 337.5 μl sterile H₂O and 37.5 μl 2.5 M CaCl₂, was precipitated by slow drop-by-drop addition of 337 μl HEPES-buffered saline pH 7.05 (42 mM HEPES, 10 mM KCl, 1.5 mM Na₂HPO₄, 1.1 mM glucose), then incubated for 30 min at RT and added to the cells. Cells were lysed after 24–48 h. The following expression plasmids were used for transfection: peGFP-N1-mPRG1, peGFP-N1-rPRG2, peGFP-N1-mPRG3, peGFP-C1-mPRG4, peGFP-N1-rPRG5 (Savaskan et al., 2004 (link); Broggini et al., 2010 (link); Velmans et al., 2013 (link); Coiro et al., 2014 (link)).

Recombinant AAV8 was used as vector for expressing murine Klotho or enhanced green fluorescent protein (GFP). The genomic constructs contained the LP-1 promoter, GFP or a codon-usage optimized sequence of murine alpha klotho (Geneart, Regensburg, Germany) with an N-terminal secretion signal originating from human CD33 (sequence: MPLLLLLPLLWAGALA) and a C-terminal V5-tag epitope (Southern et al., 1991 (link)), followed by a WPRE sequence and an SV40 polyA signal. The expression cassettes were flanked by AAV2-derived inverted terminal repeats. The recombinant AAV8-LP1-mKlotho and AAV8-LP1-eGFP vectors were prepared as previously described (Strobel et al., 2019 (link)). Briefly, human embryonic kidney cells (HEK-293H cells, Thermo Fisher Scientific) were cultured in Dulbecco's modified Eagle's medium +GlutaMAX I+10% fetal calf serum (Gibco/Thermo Fisher Scientific) and transfected as previously described (Strobel et al., 2015 (link)). AAV purification via polyethylene glycol precipitation, iodixanol gradient, ultrafiltration, and sterile filtering was conducted as described previously (Strobel et al., 2019 (link)). Genomic titers of purified AAV8 vector stocks were determined by isolation of viral DNA and subsequent qPCR analysis using primers specific for the LP-1 promoter with the following primer sequences; forward: GACCCCCTAAAATGGGCAAA. reverse: TGCCCCAGCTCCAAGGT.

HEK293-H cells (Thermo Fisher Scientific) were maintained in suspension at 37°C with 5% CO₂ in CD293 medium supplemented with 4 mM GlutaMAX (Invitrogen). The HEK293 cell line is a permanent line established from primary embryonic human kidney and transformed with sheared human adenovirus type 5 DNA. The E1A adenovirus gene is expressed in these cells to optimize protein production. HEK293-H cells were cloned from the original 293 cell line and adapted to CD293 serum-free medium for growth in suspension. Cell line identity has been authenticated by ThermoFisher Scientific, and cells were tested negative for mycoplasma by qPCR detection assay. For imaging and electrophysiology, cells were plated onto poly-L-lysine coated coverslips one day before transfection and grown in a medium containing 44% DMEM (Corning), 44% Ham’s F12 (Corning), 10% fetal bovine serum (HyClone), 2 mM glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin.