Epithelial cells that reached confluence in the four-well plates
were used for this experiment. The cells were incubated with
serum-free DF (DMEM/Ham’s F-12 1:1 (vol/vol) supplemented
with 0.1% BSA, 5 mg/ml holo-Transferrin (Sigma–Aldrich), and
2 mg/ml insulin (Sigma–Aldrich)) at 38.5, 39.5, 40.0, and
40.5 8C for 24 h. After incubation, the medium was collected in
a 1.5 ml tube containing a 1% stabilizer solution (0.3 mol/l
EDTA and 1% (w/v) acetylsalicylic acid, pH 7.3), and the
concentrations of PGE2 and PGF2a in the medium were
measured by enzyme immunoassay. The cells were collected
for the measurement of DNA amount by DNA assay.