Butyrate Modulates Collagen I in MG-63 Cells

Briefly, 1 x 10⁵ of MG-63 cells were seeded on the sterile coverslips in a 24-well plate in DMEM and 10% FBS. After 24 h, they were subjected to different concentrations of butyrate (0–16 mM) for additional 24 h. Medium was decanted, and cells were rinsed with PBS and fixed for 20 min in 4% paraformaldehyde. Cells were washed by PBS, membrane penetration with 2% Triton X-100, incubated for 20 min in 0.3% v/v H₂O₂. After washed with PBS, cells were blocked in 5% bovine serum albumin (BSA) for 1 h and then incubated in primary antibodies (against type I collagen) at room temperature overnight. After washing by PBS, cells were incubated in corresponding secondary antibody in the dark for 1 h and counterstained for 30 min with 4',6-diamidino-2-phenylindole (DAPI, 1:1000) [17 (link)]. Finally the samples were mounted and observed/photographed by an Olympus IX71 inverted microscope and DP Controller/Manager software (Olympus Corporation).