Flow cytometry analyzer lsrii

For EdU/DAPI FACS analysis, Pole4^+/+ and Pole4^−/− MEFs (passage 3) were labeled and processed using the Click-iT EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher). Cells were pulse labeled for 30 min with 10 μM EdU and fixed in 4% paraformaldehyde, before being permeabilized in PBS-Triton 0.5% and washed in 1% BSA. Cells were then resuspended in Click-iT reaction cocktail containing Alexa Fluor 488 Azide and incubated for 30 min at R.T. After being washed, cells were finally counterstained for DNA content by DAPI (1 mg/ml) and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).
For BrdU/DNA content analysis of human dermal fibroblasts derived from control or POLE1 mutant patients, cells were pulse labeled with 10 μM BrdU (SIGMA) for 30 min, washed and released or not in normal media for 4, 8, 12 or 16 hours to analyze S-phase progression. Cell were trypsinized, pelleted and fixed in ice-cold 70% ethanol. After washing, cells were resuspended in 500 mL of 2M HCl and incubated at R.T for 30 min with occasional mixing. Cells were pelleted, washed to remove excessive acid and incubated with anti-BrdU antibody for 20 min at R.T. and rabbit anti-mouse FITC-conjugated (DAKO) for 20 at R.T. in the dark. Cells were finally washed, resuspended in PBS-T containing RNase A and Propidium Iodide and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).

For indirect immunofluorescence staining, cells were seeded on coverslips and fixed in 4% paraformaldehyde. After permeabilization with 0.5% Triton X-100 (5 min on ice), coverslips were blocked in 1% BSA/PBS and incubated with the following primary antibodies diluited in 0.5% BSA/PBS: anti-H2AX phosphorylated on Ser139 (γH2AX) (Millipore), −53BP1, (Novus Biologicals), for 1h at room temperature. Coverslips were then washed 3 times in PBS and incubated with Alexa Fluor 488 goat anti-rabbit or rabbit anti-mouse antibodies (Invitrogen) for 45 min at room temperature. After DAPI counterstaining, coverslips were mounted in Glycerol/PBS (1:1) and observed with Axio Imager.M2 (ZEISS) using the Volocity 6.3 software. For EdU immunofluorescence analysis MEFs (passage 3) were labeled and processed using the Click-iT® EdU Flow Cytometry Cell Proliferation Assay (Thermo Fisher). Cells were pulse labeled for 30 min with 10 mM EdU and fixed in 4% paraformaldehyde, before being permeabilized in PBS-Triton 0.5% and washed in 1% BSA. Cells were then resuspended in Click-iT reaction cocktail containing Alexa Fluor® 488 Azide and incubated for 30 min at R.T. After being washed, cells were finally counterstained for DNA content by DAPI (1 mg/ml) and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).

Chromatin FACS analysis of endogenous RPA in Pole4^+/+ and Pole4^−/− MEFs (passage 2) and HeLa TRex POLE3 WT or ΔC mutant, as well as exogenous GFP-RPA and RFP-PCNA in U2OS GFP-RPA/RFP-PCNA were performed essentially according to Forment and Jackson, 2015 (link). Briefly, exponentially growing cells (transfected against the listed siRNAs or not) were treated or not with 2 mM hydroxyurea for 2 hr, trypsinized, harvested in media, washed in PBS and permeabilized for 10 min on ice with PBS-Triton 0.2%. After washing in 1% BSA-PBS, cells were fixed with paraformaldehyde 4% at room temperature for 15 min. Chromatin was washed again in 1% BSA-PBS and PBS, stained with DAPI for cell cytometric analysis or further processed for endogenous RPA analysis. In this case, chromatin pellet was blocked in 1% BSA for 30 min and incubated with antibody against RPA (Abcam, see key resource table) for 1 hr at room temperature at 1:200 final concentration. After wash in 1% BSA-PBS and PBS, pellet was incubated with secondary Alexa Fluor-conjugated 488 anti mouse for 30 min at room temperature, washed again in PBS and analyzed using a Flow cytometry analyzer LSRII (Becton Dickinson).