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:
 * 5) (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
 * 6) (Volume collagen to be added in ml) X (0.03) = Volume 1N NaOH to be added
 * 7) (Final Volume) - (Volume collagen to be added) - (Volume 1N NaOH to be added) = Volume PBS (or cell/medium) to be added
 * 8) Pipet into a 15 ml falcon tube in the following order:
 * 9) PBS (tube 1) or Cell (tube 2)
 * 10) NaOH (always keep tube on ice after this step)
 * 11) Vortex
 * 12) Collagen
 * 13) Vortex
 * 14) Deliver solution into each well (2 ml/well for 6-well plate).
 * 15) Place the 6-well plate in incubator for 30 min.
 * 16) Take the 6-well plate out of incubator.
 * 17) Use a glass rod to detach the gels from the wells.
 * 18) Place 6-well plates back in incubator.
 * 19) Clean hood and dispose of biohazard waste.
 * 20) Measure diameter of the gels every 24 hours.

or instead, discuss this protocol.