Migration was quantified by OD reading at 590 nm after 2-hour extraction of the crystal violet stain.
Colony formation was quantified by OD reading at 590 nm after 2-hour extraction of the crystal violet stain.
Initial studies demonstrated that TGF-β1, TGF-β2, and IGF-I could be efficiently isolated from powdered human bone using two 24 hours extractions in guanidine (4 M) EDTA (0.05 M), 0.03 M Tris buffer.
Protein extracts and Western immunoblotting experiments were performed as described previously in Koechler et al. [ 15] Strains were grown at 25°C for 24 hours (OD = 0,15 at 600 nm, corresponding to log-phase cells) and cultures were induced or not by addition of 0,66 mM As III) for 15 min, 6 hours or 8 hours before extraction.
To reduce RNA degradation the LMD procedure never exceeded one hour before extraction buffer was added to the microdissected cells.
Briefly, two liters of 24 h culture of S. boulardii were centrifuged at 3.000×g for 10 min. The supernatant was decanted and extracted with one-fifth volume of ethyl acetate with changes of ethyl acetate every half an hour of extraction.
After 1 hour, nuclear extraction and proteins were separated by acrylamide gel electrophoresis.
After 1 hour of extraction (on a magnetic stirrer), the solution was centrifuged at 4,000 rpm for 10 minutes.
Plasma was separated from whole blood by low-speed centrifugation at 1500 × g for 15 min at 4°C within the hour after extraction.
The aqueous phase, after 1 hour of extraction, containing 12.5 mg L-1 of ribitol as an internal standard, was evaporated over-night in a vacuum concentrator and the tube was then returned to the -80°C freezer until use.
Results: Maximum postoperative pain was recorded 6 hours after extraction, with peak inflammation after 24 hours.
