Leibowitz l 15 medium

Immortalized baby mouse kidney (iBMK) cells were cultured in 12-well plates on 18-mm diameter No.1 glass coverslips in complete growth medium consisting of high glucose DMEM (Invitrogen cat. #11965) supplemented with 10% fetal bovine serum and 100 U/mL of penicillin and $100\mu \mathrm{g}/\mathrm{mL}$ of streptomycin (1% pen/strep). The cell cultures were maintained in an incubator at 38°C with an 8.5% ${\mathrm{CO}}_2$ in air atmosphere as previously described.³² (link) Prior to imaging, the cells were transfected with the FRET constructs using lipofectamine LTX (Invitrogen) following the manufacturer’s protocol. We used $1.25\mu \mathrm{g}$ DNA, $1.25\mu \mathrm{L}$ PLUS reagent, and $3\mu \mathrm{L}$ lipofectamine reagent in $125\mu \mathrm{L}$ OptiMEM, for each culture well with cells grown on an 18-mm-diameter coverslip. The cells were incubated with the transfection mixture for 1 to 3 h in OptiMEM medium (Invitrogen) after which the OptiMEM medium was replaced with the complete growth medium described above. The cells were imaged 24 to 72 h after transfection. During imaging, the cells were placed in an imaging medium consisting of a non- ${\mathrm{CO}}_2$ -dependent buffered Leibowitz L15 medium (Invitrogen) supplemented with 10% FBS and 1% pen/strep. The coverslip with attached cells was mounted on a homemade steel plate sealed with vacuum grease and VALAP (1:1:1 ratio of vaseline, lanolin, and paraffin wax). The cells were imaged at room temperature and room air.