Lipofecatime 3000

Stable cell lines were generated using either human embryonic kidney (HEK) HEK293 (ATCC, Manassas, VA) or HEK293-6E (National Research Council, Canada) cells^{25 (link)}. Briefly, cells were transiently transfected with 2CS, 1CS, or G32R null-biosensor plasmids using Lipofecatime 3000 or 293fectin (Thermo Fisher Scientific). Flow cytometry was used to select and enrich for the population of cells expressing respective biosensors. Stable HEK293 cell lines were maintained in phenol red-free DMEM media (Gibco, Waltham, MA) supplemented with 2 mM GlutaMAX (Gibco, Waltham, MA), 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1 IU/mL penicillin/streptomycin (Gibco, Waltham, MA), and 250 μg/mL G418 (Fisher Scientific). Stable HEK293-6E cell lines were maintained in F17 media (Sigma Aldrich) supplemented with Kolliphor p188 (Sigma Aldrich, St. Louis, MO), 200 nM/mL GlutaMAX, and either 1 μg/mL puromycin (Invitrogen, Carlsbad, CA) or 2 μg/mL blasticidin (Goldbio) as a selection antibiotic. All cell lines were grown at 37 °C with 5% CO₂.

Stable cell lines were generated using either HEK293 (ATCC, Manassas, VA) or HEK293–6E (National Research Council, Canada) cells^{20 (link)}. Briefly, cells were transiently transfected with 2CS, 1CS, or G32R null biosensor plasmids using Lipofecatime 3000 or 293fectin (Thermo Fisher Scientific). Flow cytometry was used to select and enrich for the population of cells expressing respective biosensors. Stable HEK293 cell lines were maintained in phenol red-free DMEM media (Gibco, Waltham, MA) supplemented with 2 mM GlutaMAX (Gibco, Waltham, MA), 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1 IU/mL penicillin/streptomycin (Gibco, Waltham, MA), and 250μg/mL G418 (Fisher Scientific). Stable HEK293–6E cell lines were maintained in F17 media (Sigma Aldrich) supplemented with Kolliphor p188 (Sigma Aldrich, St. Louis, MO), 200 nM/mL GlutaMAX, and either 1 μg/mL puromycin (Invitrogen, Carlsbad, CA) or 2μg/mL blasticidin (Goldbio) as a selection antibiotic. All cell lines were grown at 37°C with 5% CO₂.