Jacobs:Protocol Collagen Gel Contraction 2
- 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
- Microfuge tube
- Cell counter
- Trypan blue
- 15, 50 ml Falcon tubes
- Waste beaker
- Water bath
- Biohazard bag
- 70% ethanol
- Markers, notebooks, gloves
- 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
- Prepare hood. Place materials inside hood.
- Subculture 3T3 cells so that 25,000 cells per cm2 in differentiation media can be obtained.
- Place the collagen and NaOH on ice. (always keep it on ice)
- Make following calculations:
- (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
- (Volume collagen to be added in ml) X (0.03) = Volume 1N NaOH to be added
- (Final Volume) - (Volume collagen to be added) - (Volume 1N NaOH to be added) = Volume PBS (or cell/medium) to be added
- Pipet into a 15 ml falcon tube in the following order:
- PBS (tube 1) or Cell (tube 2)
- NaOH (always keep tube on ice after this step)
- Deliver solution into each well (2 ml/well for 6-well plate).
- Place the 6-well plate in incubator for 30 min.
- Take the 6-well plate out of incubator.
- Use a glass rod to detach the gels from the wells.
- Place 6-well plates back in incubator.
- Clean hood and dispose of biohazard waste.
- Measure diameter of the gels every 24 hours.
or instead, discuss this protocol.