Cell culture. All animal procedures were approved by the University of California’s institutional IACUC protocol, in accordance with NIH policy. Neurons were prepared from C57BL/6 mice. Briefly, hippocampi were dissected from postnatal day 1 mice, dissociated using papain and trituration, and plated MEA Recordings. Extracellular recordings were obtained using a Multi Channel Systems (MCS, Reutlingen, Germany) MEA2100 system and a 120count multielectrode array with 100 μm interelectrode spacing and 30 μm electrode diameter, at a sampling frequency of 20 kHz.
Cell culture. Wildtype HepG2 cells were maintained in DMEM with 5 mM glucose, 3.7 g L-1 NaHCO3, 1 mM sodium pyruvate and amino acids, supplemented with 3 mM glutamine, and 10% FBS. The cells were kept at 37 °C and 5% CO2. To test the individual and combined effects of glucose depletion and fatty acid stimulation on the free CoA level and acyl-CoA profile, the cells were incubated for 24 h in two different conditions. In condition 1 the cells were cultured in DMEM (5 mM glucose, 1 mM pyruvate supplemented with 3 mM glutamine and 10% FBS) with additional supplements 2 mM L-carnitine and 0.5 mM BSA-bound palmitate. Condition 2 was with glucose-free DMEM (no glucose, no glutamine, no pyruvate, 10% FBS) supplemented with 2 mM L-carnitine and 0.5 mM BSA-bound palmitate. After 24 h, the cells were washed twice with ice-cold PBS and harvested for further analysis. Condition 1 cells were "supplemented cells" with multiple carbon sources, while condition 2 cells were "starved cells" with fewer carbon sources.
Cell culture. 1.1. [*] vials of the primary and back-up cell line which produce the BR96 antibody [*] Confidential Treatment Requested
1.2. List of raw materials and specifications (e.g. vendor, catalog number)
1.3. Process protocols (e.g. media formulations, thaw and expansion protocol, final production tank protocol)
1.4. Historical process data (e.g. thaw and expansion data, in-process data from final production tank)
1.5. Cell-line stability data (as it becomes available)
Cell culture. JIMT-1 cells were purchased from DSMZ (Braunschweig, Germany); all other cell lines were purchased from American Type Culture Collection (Manassas, VA). HCC1419 and HCC1954 cells were maintained in RPMI with 10% fetal bovine serum (FBS); MDA- MB-361 was maintained in RPMI with 20% FBS; JIMT-1, BT474, and MDA-MB-453 were maintained in DMEM with 10% FBS; all cells were maintained in 1% penicillin/ streptomycin and cultured in humidified incubators at 37°C with 5% CO2.
Cell culture. Unless otherwise stated, solutions referred to in the text were made according to table 2.1. All solutions were made in a class II microbiological safety cabinet (MSC).
Cell culture. XXX-000X/00, X0x, xxx XXX-0X0 cells were acquired from ATCC (Manassas, VA) and maintained in DMEM (Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humid, 5% CO2, 37°C incubator. HEK293T and NIH-3T3 cells were transfected using Mirus TransITLT1 (Madison, WI) per the manufacturer’s protocol. N2a cells were transfected using Lipofectamine LTX (Thermo Xxxxxx Scientific) per the manufacturer’s protocol.
