Primary muscle cells from in-utero undernourished mice. Mus musculus

ICR mice, age 6-8 weeks.(Harlan, Indianapolis, IN, USA). Housed individually with controlled temperature, humidity, and light-dark cycle (0600h – 1800h). Pregnant mice were housed individually with ad libitum access to standard rodent chow (T.2018, Harlan Teklad, Indianapolis, IN, USA). On day 12.5 of pregnancy, dams were randomly assigned to either a control or undernutrition group. In the undernutrition group, food was restricted to 50% that of gestational day -matched controls for the remainder of pregnancy. After delivery, mothers were given ad libitum access to chow and 24 hours after birth, litters were equalized to eight. We studied mice from 2 groups: in utero undernourished offpring (U) and control offspring (C). Primary muscle cells were isolated from the quadriceps of U and C at 3 weeks old. Isolated primary muscle cells were grown on Matrigel coated flasks in low glucose (5.5 mmol/L) Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 20% FBS, 1X antibiotic-antimycotic, 2.5μg/ml gentamycin and 2.5ng/ml basic fibroblast growth factor. When cells reached approximately 90% confluency, they were differentiated in low glucose DMEM supplemented with 2% FBS, 1X antibiotic-antimycotic, and 2.5μg/ml gentamycin. Other cells were cultured overnight in substrate limited medium (glucose and glutamine free DMEM supplemented with 0.5mM glucose, 1mM glutamine, 0.5mM carnitine and 1% (v/v) FBS; pH 7.4). Overall design: RNA expression analysis of primary muscle cells from in-utero undernourished mice, cultured in substrate limited medium and standard medium.