To produce lentivirus, pLenti emGFP-Slc37a2, and pHIV-PV-VSVG (kind gift from Dr. Scott A. Fisher, The University of Western Australia) packaging plasmid were co-transfected into HEK293FT cells (R70007, Thermo Fisher) using TransIT-Lenti transfection reagent (Mirus) according to manufacturer’s instructions. 18 h post-transfection, media containing the transfection reagent was removed and replaced with fresh, high-BSA DMEM (Gibco) containing 10% FBS (Gibco) and 0.1 g/L bovine serum albumin (Sigma-Aldrich). Culture media containing lentivirus were collected 48–72 h post-transfection and concentrated using a lentivirus concentration solution (Origene) according to the manufacturer’s instructions. The lentiviral pellet obtained post concentration was resuspended in cold, sterile PBS, aliquoted, and stored at −80 °C until required.
Prior to transduction, lentiviral particles were titrated using the qPCR lentivirus titration kit (Applied Biological Materials Inc.) according to the manufacturer’s instructions. BMMs were transduced with lentiviral particles in the presence of 20 µg/ml protamine sulfate (Sigma-Aldrich) 24 h after the first RANKL (10 ng/ml, R&D Systems) stimulation of BMMs.
Prior to transduction, lentiviral particles were titrated using the qPCR lentivirus titration kit (Applied Biological Materials Inc.) according to the manufacturer’s instructions. BMMs were transduced with lentiviral particles in the presence of 20 µg/ml protamine sulfate (Sigma-Aldrich) 24 h after the first RANKL (10 ng/ml, R&D Systems) stimulation of BMMs.