To assess the effects of SIRT1 on cell contraction, fibroblasts were seeded in type I collagen gels (BD Biosciences) that were then incubated in medium with or without TGFβ2 (10 ng/ml) and resveratrol for up to 48 hours (15 (link)). At the end of the incubation periods, gel diameters were determined. Modulation of cell migration by SIRT1 was evaluated by in vitro wounding assays (15 (link)). Briefly, confluent monolayers of fibroblasts were incubated in serum-free medium with resveratrol for 12 hours in the presence of 10 µg/ml mitomycin C (Sigma), and scratch wounds were inflicted using standard p1000 pipette tips. Cell migration was then monitored by phase-contrast microscopy for up to 48 hours. Gap width was determined at 3 different sites per sample at indicated intervals.
Protocol detail
SIRT1 Modulates Fibroblast Contraction and Migration
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Assays
Cell migration
Fibroblasts
Mitomycin c
Phase contrast microscopy
Resveratrol
Serum
Sirt1
Type i collagen
Wounds
Corresponding Organization :
Other organizations : Northwestern University, Fudan University
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Variable analysis
independent variables
- Presence or absence of TGFβ2 (10 ng/ml)
- Presence or absence of resveratrol
- Gel diameters
- Cell migration
- Type I collagen gels (BD Biosciences)
- Serum-free medium
- Mitomycin C (10 µg/ml)
- Positive control: Fibroblasts incubated in medium with TGFβ2 (10 ng/ml)
- Negative control: Fibroblasts incubated in medium without TGFβ2 and resveratrol
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