Jacobs:Protocol Adipocyte Differentiation

Overview

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

Materials

• 3T3 Fibroblasts (ATCC)
• 0.25% Trypsin/0.03% EDTA (Gibco #25200-056)
• Complete medium (Growth medium)

450 ml DMEM (Gibco #11995065) + 50 ml Calf Serum (Gibco #16010159)

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

Notes

Used in Stanford for Tissue Engineering Lab Course (ME385B)

Contact

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

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