Jacobs:Protocol Collagen Gel Contraction 2

Materials

• Complete medium = DMEM supplemented with 10% bovine calf serum
• 0.25% Trypsin-0.03% EDTA
• 6-well plates
• Ice, ice bucket
• Pasteur pipets
• Pipetter, Sterile pipette tips
• Pipet aid, Serological pipets
• Hemocytometer
• Microfuge tube
• Cell counter
• Trypan blue
• Timer
• 15, 50 ml Falcon tubes
• Waste beaker
• Centrifuge
• Water bath
• Biohazard bag
• 70% ethanol
• Markers, notebooks, gloves

Procedure

Differentiation Media

• 3T3 growth media (DMEM-Gibco 11995065 + 5% CS-Gibco 16010159)
• 200uM indomethacin (MP Biomedicals-190217)
• 10ug/mL insulin (Sigma-I6634) in pH 2.0 H2O
• 0.5mM IBMX (MP Biomedicals-195262) in DMSO
• 1uM dexamethasone (MP Biomedicals-194561) in DMSO

1. Prepare hood. Place materials inside hood.
2. Subculture 3T3 cells so that 25,000 cells per cm2 in differentiation media can be obtained.
3. Place the collagen and NaOH on ice. (always keep it on ice)
4. Make following calculations:
   1. (Final Volume) X (Final collagen concentration in mg/ml) / (Concentration in bottle) = Volume collagen to be added Note: Final volume = Number of wells X 2 ml (for 6-well plate); Final collagen concentration = 1.25 mg/ml; Concentration in bottle = 4.27 mg/ml
   2. (Volume collagen to be added in ml) X (0.03) = Volume 1N NaOH to be added
   3. (Final Volume) - (Volume collagen to be added) - (Volume 1N NaOH to be added) = Volume PBS (or cell/medium) to be added
5. Pipet into a 15 ml falcon tube in the following order:
   1. PBS (tube 1) or Cell (tube 2)
   2. NaOH (always keep tube on ice after this step)
   3. Vortex
   4. Collagen
   5. Vortex
6. Deliver solution into each well (2 ml/well for 6-well plate).
7. Place the 6-well plate in incubator for 30 min.
8. Take the 6-well plate out of incubator.
9. Use a glass rod to detach the gels from the wells.
10. Place 6-well plates back in incubator.
11. Clean hood and dispose of biohazard waste.
12. Measure diameter of the gels every 24 hours.

or instead, discuss this protocol.