Cells were plated in 24-well plates and counted 24 hours later (time 0), to determine seeding efficiency. OGF or other compounds were added at time 0, and media and compounds were replaced daily. All drugs were prepared in sterile water, and dilutions represent final concentrations of the compounds. An equivalent volume of sterile water (vehicle) was added to control wells. At designated times, the cells were harvested, stained with trypan blue, and counted with a hemacytometer. At least two aliquots per well of at least two wells per treatment per time point were sampled. Three to five independent assays were performed for each of the experiments (i.e., dose–response, growth curves, receptor mediation, and reversibility).
To determine the specific opioid peptide involved in growth regulation, we added a variety of endogenous peptides and synthetic compounds (10−6 M) to the RTCFs for 72 hours, including [DAla2, MePhe4, Glycol5]-enkephalin (DAMGO), morphine, [D-Pen2,5]-enkephalin (DPDPE), [D-Ala2, D-Leu5]-enkephalin (DADLE), dynorphin A, endomorphin-1, U-69593, and β-endorphin, as well as the opioid antagonist naltrexone (NTX). An equivalent volume of sterile water served as the control. Drug or vehicle and medium were changed daily. Cell proliferation was then evaluated (Promega Cell Titer 96 Aqueous One Solution Cell Proliferation Assay; Promega Corp., Madison, WI). Absorbance was recorded at 290 nm with a 750-nm filter on a plate reader (Bio-Rad, Hercules, CA). The plate consisted of 10 wells per treatment and the mean of those absorbencies was plotted. Three independent experiments were performed.