For EdU FACS, cells were treated with 10 μM EdU for 10 min, trypsinized, washed with PBS, and fixed with cold 70% ethanol on ice for 30 min to overnight. Cells were washed with PBS, and EdU staining was performed by using the EdU Click-iT kit (Thermofisher, # C10632), according to the manufacturer’s instructions. For DNA staining, we used 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) or FxCycle™ PI/RNase Staining Solution (Thermofisher, #F10797).
Chromatin association of MCM was assessed essentially as described46 (link): after trypsinization and PBS wash, cells were extracted with CSK buffer (10 mM PIPES pH 7.0, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, protease inhibitors (Pierce #A32953), fixed with 4% paraformaldehyde, and blocked with 5% BSA followed by immunostaining with anti-MCM7 antibody (Santa Cruz, #sc-9966) at 1:200 dilution. 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) was used for DNA staining.
Flow cytometry was performed on an FACSCalibur flow cytometer, and data were analyzed by using FCSalyzer software. GraphPad Prism 9 was used for statistical analyses.
Chromatin association of MCM was assessed essentially as described46 (link): after trypsinization and PBS wash, cells were extracted with CSK buffer (10 mM PIPES pH 7.0, 300 mM Sucrose, 100 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100, protease inhibitors (Pierce #A32953), fixed with 4% paraformaldehyde, and blocked with 5% BSA followed by immunostaining with anti-MCM7 antibody (Santa Cruz, #sc-9966) at 1:200 dilution. 7-AAD (7-Aminoactinomycin D) (Thermofisher, # A1310) was used for DNA staining.
Flow cytometry was performed on an FACSCalibur flow cytometer, and data were analyzed by using FCSalyzer software. GraphPad Prism 9 was used for statistical analyses.
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