Facsaria 3 sorter instrument

For BrdU labeling experiment, BrdU (final 20uM) was added to the medium for 2 h before cells were collected and cells were harvested and fixed in 70% ethanol. The cells were permeabilized with 2 N HCl–0.5% Triton X-100, neutralized with 0.1 M sodium tetraborate, stained with monoclonal anti-BrdU (BD Biosciences), and then with anti-mouse IgG F(ab)2-FITC (Sigma), and counterstained with PBS-7-AAD-RNase A. Measurements of the immunofluorescent cells were performed with a FACS Calibur analyzer (BD) and analyzed with FCSexpress software.
Flow cytometric analysis was performed on a BD FACSAria™ III sorter instrument equipped with BD FACSDiva™ 7.0 software (BD Biosciences, New Jersey, USA). FITC 490nm fluorescence was acquired in logarithmic amplification in FL1 and 7-AAD 650nm fluorescence was acquired in linear amplification in FL3.

For the bromodeoxyuridine (BrdU) labeling experiment, BrdU (Final 20 μM) was added to the medium 2 h before collection of cells. Cells were then harvested and fixed in 70% ethanol. The cells were permeabilized with 2 N HCl–0.5% Triton X-100, neutralized with 0.1 M sodium tetraborate, stained with monoclonal anti-BrdU (BD Biosciences), and then with anti-mouse IgG F(ab)2-FITC (Sigma), and counterstained with PBS-7-AAD-RNase A. Flow cytometric analysis was performed on a BD FACSAria™ III sorter instrument equipped with BD FACSDiva™ 7.0 software (BD Biosciences, New Jersey, USA). FITC 490 nm fluorescence was acquired in logarithmic amplification in FL1 and 7-AAD 650 nm fluorescence was acquired in linear amplification in FL3. Cell cycle analysis was done using Cytomics™ FC500 Flow Cytometry CXP 2.0.