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In this study, the aerobic growth capabilities of iJN746 in iM9 medium substituted with different carbon sources were determined qualitatively (Table 2) and quantitatively (Table 3).
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On day 2 (d2), the serum containing medium was substituted with serum replacement medium (KO) and this resulted in initial proliferation and a gradual switching to differentiation.
After 1 h incubation, the medium was substituted with ordinary growth medium.
For these measurements, cultures were washed and the medium was substituted with HEPES-buffered saline medium before stimulation.
The next day, the medium was substituted with DMEM/F12 containing 0.1% FBS with or without siRNA to render the cells quiescent.
For cytoplasmic pH measurement, the cultures were resuspended at OD600 0.4 in buffered M63 medium [36] substituted with 0.2% casein hydrolysate (M63A) adjusted to pH 7.5 (5 mM HOMOPIPES).
The medium was substituted with Knockout-DMEM (Kon on d2 that contained all the additives and Knockout serum replacement (Invitrogen) in place of FBS and was supplemented with (KR) or without 100 nM all-trans retinoic acid (RA).
For fusion assays, the medium was substituted with DMEM containing 5% horse serum and 100 U/ml penicillin and 100 µg/ml streptomycin.
To differentiate L6 myoblasts into myotubes, the culture medium was substituted with DMEM plus 2% horse serum after the L6 myoblast cells became confluent.
Growth medium was then substituted with fresh medium containing the drugs to be tested.
The AA-free medium was then substituted with the AA-supplemented medium.
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