Cell Proliferation Assay Using Tritiated Thymidine

³H-thymidine incorporation assays were performed as reported early [9 (link), 23 (link)]. CFb were plated at equal densities in 24-well plates (6 wells per condition) and allowed to adhere overnight. The cells were stimulated by the addition of PDGF (10 ng/ml) for 18 h and then incubated with 2 μCi/ml ³H-thymidine (6.7 Ci/mmol; Amersham, Arlington Heights, IL) for 4 h. The following protocol was used to measure ³H-thymidine incorporation: cells were 1) rinsed two times in cold PBS, 2) 10% trichloroacetic acid added for 30 min at 4°C, 3) washed in cold 100% ethanol, 4) solubilized in 0.1 N NaOH for 30 min at 65°C, and 5) radioactivity measured in a scintillation counter.