2.5 | LF Cell Culture
The LF specimens obtained from patients during surgery were washed 3
times with phosphate-buffered saline (PBS, Gibco), minced into into
small fragments of approximately 0.5mm3 and digested
using 0.2% type I collagenase (Gibco) at 37℃ for 1.5h. Subsequently,
the digested pieces were washed with Dulbecco’s modified Eagle’s medium
(DMEM, Gibco) and cultured with DMEM supplemented with 10% fetal bovine
serum (Sigma), 100 U/ml penicillin and 100 pg/ml streptomycin (Sigma) in
a 5% CO2 humidified incubator at 37℃. The cell-culture
medium was changed every two to three days. The second passage of cells
was used for the subsequent experiments.
The expression levels of collagen I and Ⅲ were detected by
immunostaining and observed under a confocal microscope (XTL3230-DIC,
Shanghai, China) to identify the cell type. The second passage of LF
cells was cultured and stimulated with with different concentrations of
recombinant human WISP-1 ranging from 0 to 150ng/ml for 24 h. For
Hedgehog signaling inhibition experiments, 106 cells
were seeded in each well of 6-well plates. Then, the indicated dose
(5μM) of inhibitor (cyclopamine, Sigma, USA) without cytotoxicity were
added in the culture medium for 24 h. Finally, cells were collected for
subsequent experiments.