Table 1.
Overview of the Cell Culture and Assay Protocols
| A. 3T3L1 Adipocytes | |||
|---|---|---|---|
| Step | Parameter | Value | Description |
| 1 | Cell maintenance | 75 cm2 flask | Split 1:2, weekly, into flasks for maintaining the cell line and flasks for adipogenesis. |
| 2 | Adipogenesis | 75 cm2 flask | Medium supplemented with 0.5 mM IBMX, 0.25 μM dexamethasone, and 1 μg/mL insulin for 3 days. |
| 3 | Seeding for HTS | 96-well dish | Seed 30,000 to 60,000 differentiated adipocytes/well in maintenance medium. |
| 4 | High-content assay | 96-well dish | 2–5 days postplating, cells were exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and protein. |
| B. Human Subcutaneous Adipocytes | |||
|---|---|---|---|
| Step | Parameter | Value | Description |
| 1 | Adipogenesis | 96-well dish | Preadipocytes (passage 2) were seeded at 13,000 cells/well and exposed to 300 nM rosiglitazone for 17 days. |
| 2 | High-content assay | 96-well dish | Cells were exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and protein. |
(A) 1
To maintain the 3T3L1 line, cells were cultured before reaching confluence. To prepare for adipogenesis, cells were cultured 2–3 days after achieving confluence to insure they were postmitotic.
3
Precoating of the 96-well glass-bottomed dishes with either gelatin (cross linked with glutaraldehyde) or Matrigel (diluted 1/50) is necessary to insure adipocyte adherence. A large number of adipocytes were plated per well, as confluent cell cultures are best analyzed by CyteSeer®.
(B) 2
Experiments were performed in the absence of rosiglitazone (cells were switched to rosiglitazone-free medium, 2 h before experimentation).
IBMX, 3-isobutyl-1-methylxanthine; HTS, high-throughput screening.