Jacobs:Protocol Adipocyte Differentiation

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Contents

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)

References

Contact

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


or instead, discuss this protocol.

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