Ultraculture serum free media

Human kidney 293T cells were purchased from Alstem Inc (Richmond, CA). Human liver HepG2 cells were purchased from ATCC (Manassas, VA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) and Opti-MEM 1 × reduced serum media were purchased from Gibco (Billings, Montana). Ultraculture serum free media was purchased from Lonza (Hayward, CA). Fetal Bovine Serum (FBS) was purchased from HyClone Laboratories (Logan, UT). Polyethylenimine (PEI, Product No. 18978) was purchased from Millipore Sigma. Recombinant Gaussia princeps luciferase (CAT #321-100) was purchased from Nanolight Technologies (Pinetop, AZ). ExoQuick TC was purchased from System Biosciences (Palo Alto, CA). Brefeldin A (BFA) was purchased from eBioscience (San Diego, CA). Pierce™ Gaussia Luciferase Glow Assay Kit was purchased from Thermo Scientific (Waltham, MA).

Lentiviral constructs expressing shRNA to rat IP6K1 (IP6K1 shRNA, 5′-TCAACCTGGTAGCCTACCCTT-3′) and IP6K3 (5′-CCATCTCGGCCTGGTTGCCAA-3′, sequences provided by A. Chakraboty, St. Louis U) (Rao et al., 2014b (link); Fu et al., 2015 (link)) were made in pFHUBW vector (gift of Roger Nicoll, UCSF), a variant of pFHUGW containing the monomeric blue fluorescent protein mTagBFP in place of GFP (Lois et al., 2002 (link)). The resulting pFHUBW vector was cotransfected along with two packaging plasmids (pVSV-G and psPAX2) into HEK293T cells using FuGENE HD (Promega), as previously described (Foss et al., 2013 (link)). HEK293T cells were grown in UltraCULTURE serum-free media (Lonza) and supplemented with 1 mM sodium pyruvate, 0.075% bicarbonate and 2 mM GlutaMax. At approximately 16 h after transfection, 10 μM sodium butyrate was added to the culture media, and at 40 h after transfection, the culture media was collected, and viral particles concentrated by centrifugation through a 20% sucrose/PBS gradient at 80,000 × g for 2 h at 4°C. Viral particles were resuspended in neuronal culture media supplemented with 4 μg/ml Polybrene (hexadimethrine bromide) (Zhang et al., 2010 (link)).