100 mm cell culture dish

Colony forming
assays were performed,
as previously described.^{45 (link)} Cells were suspended
in IMDM media containing 2% fetal calf serum (FCS; Cytiva) at a concentration
of 1 × 10⁵ cells/mL. In a 15 mL tube, 300 μL
of cell suspension was added to 3 mL of MethoCult GF H4434 medium
(Stemcell Technologies; Vancouver, BC) containing 1% methylcellulose
in IMDM, 30% FCS, 1% bovine serum albumin (BSA; Sigma-Aldrich), 3
U/mL recombinant human erythropoietin, 100 μM 2-mercaptoethanol,
2 mM l-glutamine, 50 ng/mL recombinant human stem cell factor,
10 ng/mL recombinant human granulocyte macrophage-colony stimulating
factor, and 10 ng/mL recombinant human IL-3. The cells were plated
in a 35 mm cell culture dish (Corning; Tewksbury, MA) at a concentration
of 10⁴ cells/dish using a 5 mL syringe with a blunt tip
needle (Covidien; Minneapolis, MN). Replicate dishes of each treatment
were stored in a 100 mm cell culture dish (Corning) with an additional
uncapped 35 mm dish containing distilled water to control humidity.
The plates were incubated for 7–14 days at 37 °C with
5% CO₂ and 95% humidity. The colonies were counted on an
inverted microscope; clusters of 10 or more cells were counted as
one colony.

Detailed protocol for maintenance, cryopreservation, and differentiation of white and brown preadipocytes are outlined in a different study [111 (link)]. Briefly, for culturing preadipocytes, cells were grown in DMEM medium (Corning, 10-017-CV) supplemented with 10% vol/vol FBS and containing 1% vol/vol Penicillin-Streptomycin (Gibco). Cell culture was maintained at 37 °C in a humidified incubator containing 5% vol/vol CO2. 80% confluent cells were passaged using 0.25% trypsin with 0.1% EDTA (Gibco, 25,200-056) for a 1:3 split in a new 100 mm cell culture dish (Corning).
Prior to adipogenic differentiation, white preadipocytes were allowed to grow up to 100% confluence in a 100 mm cell culture dish (Corning). After 48 h at 100% confluence, growth media was replaced with adipogenic induction media every 48 h for the next 20 days. Induction media was prepared by adding 1 mL FBS, 500 μl Penicillin-Streptomycin, 15 μl human Insulin (0.5 μM, Sigma–Aldrich, I2643-50 MG), 10 μl T3 (2 nM, Sigma–Aldrich,T6397-100 MG), 50 μl Biotin (33 μM, Sigma–Aldrich, B4639-100 MG), 100 μl Pantothenate (17 μM, Sigma–Aldrich, P5155-100G), 1 μl Dexamethasone (0.1 μM, Sigma–Aldrich, D2915-100 MG), 500 μl IBMX (500 μM, Sigma–Aldrich, I7018-100 mg), and 12.5 μl Indomethacin (30 μM, Sigma–Aldrich, I7378-5G) to 48.5 mL DMEM medium and sterile filter.

The BEL-7402 cells, HeLa cells, and MIHA cells were cultured on 100 mm cell culture dish (Corning) in DMEM-HG supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Sodium pyruvate was added to the culture media when culturing MIHA cells. Cells were grown in a humidified incubator at 37°C and 5% CO₂. After reaching confluence, cells in cell culture dish were trypsinized to suspend in aqueous environment. Trypsin was removed by centrifuging the cell sample at 250 g for 5 minutes. Glutaraldehyde was added to re-suspend the cells and fix the cells. Glutaraldehyde was removed by centrifuging the cell sample by 250 g for 5 minutes. Phosphate buffered saline (PBS) was added to re-suspend the cells which are then loaded to the microfluidic channel for ATOM experiments.