We performed the
differentiation assays with 3T3-L1 cells in accordance with a previously
described method.28 (link) In brief, an experiment
consists of 3 days predifferentiation (1 day seeding and 2 days to
allow cells to enter the resting state), followed by an 8 days differentiation
window (2 days induction of differentiation and 6 days maintenance).
Subconfluent cells of passage 10 were trypsinized and counted with
a flow cytometer (NovoCyte, Acea Biosciences). 15,000 cells well–1 were seeded in 200 μL of preadipocyte medium
(PAM: DMEM-high supplemented with 10% bovine calf serum and 1% penicillin/streptomycin)
into 96-well black, clear-bottom tissue culture plates (655,090, Greiner
Bio-One) and incubated at 37 °C and 5% CO2. After
24 h, we checked that the cells had reached confluency, replaced the
medium with 200 μL fresh PAM well–1, and cultured
the cells for another 48 h to initiate growth arrest. We included
a preadipocyte control (undifferentiated cells) on every plate which
we kept cultivated in PAM, while the rest of the cells were differentiated
as described below.
differentiation assays with 3T3-L1 cells in accordance with a previously
described method.28 (link) In brief, an experiment
consists of 3 days predifferentiation (1 day seeding and 2 days to
allow cells to enter the resting state), followed by an 8 days differentiation
window (2 days induction of differentiation and 6 days maintenance).
Subconfluent cells of passage 10 were trypsinized and counted with
a flow cytometer (NovoCyte, Acea Biosciences). 15,000 cells well–1 were seeded in 200 μL of preadipocyte medium
(PAM: DMEM-high supplemented with 10% bovine calf serum and 1% penicillin/streptomycin)
into 96-well black, clear-bottom tissue culture plates (655,090, Greiner
Bio-One) and incubated at 37 °C and 5% CO2. After
24 h, we checked that the cells had reached confluency, replaced the
medium with 200 μL fresh PAM well–1, and cultured
the cells for another 48 h to initiate growth arrest. We included
a preadipocyte control (undifferentiated cells) on every plate which
we kept cultivated in PAM, while the rest of the cells were differentiated
as described below.
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