These four types of outsourcing strategies are briefly differentiated below: 1.
Briefly, differentiated brown adipocytes were treated with insulin for 30 minutes after which 2-deoxy-[3H] glucose (0.5 µCi/ml, final concentration) was added for an additional 3 minutes.
Briefly, differentiated cells were pre-treated for 24 hr with 50 µg/ml SurR9-C84A, 50 µg/ml ascorbic acid and then treated with 300 µM H2O2 for 3 hr.
Briefly, differentiated U937 cells (1×105 cells per well in 96-well plates (Falcon 353072; Becton Dickinson, Franklin Lakes, NJ, U.S.A).) or A. polyphaga (1×105 cells per well in 96-well plates) were infected with L. pneumophila strains cultured in the post-exponential phase at a multiplicity of infection (MOI) of 0.5 or 10, respectively.
Lipid accumulation in adipocytes was visualized by staining with Oil Red O. Briefly, differentiated adipocytes were washed with phosphate buffered saline (PBS) and fixed with 10% formaldehyde in PBS for 4 min. After two washes with water, cells were stained for 1 2 hrs with Oil Red O working solution (0.3% (wt/vol) in 60% isopropanol).
Briefly, differentiated adipocytes were starved in glucose-deprived DMEM for 1 h.
Briefly, differentiated 3T3-L1 adipocytes were treated with insulin for 30 minutes after which 2-deoxy-[H] glucose (0.5 μCi/ml, final concentration) was added for additional 3 minutes.
Slides were then briefly differentiated in 70% ethanol and dehydrated through an ethanol series before xylene clearing and mounting with Histomount (National Diagnostics).
Briefly, differentiated primary wild-type myotubes were loaded with the calcium indicator dye fura-2, and the cells were imaged using an intensified charge-coupled device camera.
Briefly, differentiated adipocytes grown on six-well plate were fixed in 1X fixative solution containing 2% formaldehyde and 2% glutaraldehyde for 10 minutes, and then stained overnight at 37°C with the β-galactosidase staining solution at pH 6.0 for 18 hours.
