Jacobs:Protocol Adipocyte Differentiation

Overview
This protocol will induce adipocytic differentiation in 3T3 Fibroblasts that can be visualized by Oil Red O staining.

Materials
450 ml DMEM (Gibco #11995065) + 50 ml Calf Serum (Gibco #16010159)
 * 3T3 Fibroblasts (ATCC)
 * 0.25% Trypsin/0.03% EDTA (Gibco #25200-056)
 * Complete medium (Growth medium)
 * PBS
 * Water bath
 * Tissue culture dish (for Western Blot)
 * 6 well plate (for Oil Red O Staining)
 * Serological Pipets/Pipet aid
 * Pipette tips/Pipetter
 * 15 ml, 50 ml Falcon tubes
 * Trypan blue
 * Hemocytometer, coverslip
 * Microcentrifuge tubes
 * Centrifuge
 * Cell counter
 * Timer
 * Waste beaker
 * 70% ethanol
 * Kimwipes
 * Markers
 * Gloves

Solutions

 * Adipogenic Media
 * Growth medium (DMEM supplemented with 10% CS)
 * 200um indomethacin
 * 10ug/mL insulin
 * 0.5mM IBMX
 * 1uM dexamethasone
 * Growth Media + Insulin
 * Growth medium (DMEM supplemented with 10% CS)
 * 10ug/mL insulin

Procedure

 * 1) Day 1
 * 2) Heat complete medium, trypsin/EDTA, and PBS to 37C in water bath
 * 3) Subculture 3T3 cells with the following concentrations: (25,000cells/cm2)
 * 4) *6 well plate: 2.27x105cells/well in 6ml media
 * 5) *tissue culture dish: 1.42x106cells/dish in 10ml media
 * 6) Place cells in incubator for 1-2hr
 * 7) Replace growth media with Adipogenic Media
 * 8) Day 3
 * 9) *Replace Adipogenic Media
 * 10) Day 4
 * 11) *Change from Adipogenic Media to Growth Media + Insulin (adipocyte maintenance)
 * 12) Day 6
 * 13) *Replace Growth Media + Insulin (adipocyte maintenance)

Contact

 * Originally prepared by CRJ-EJC, last updated 2/21/07

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