MCF10A cells were grown in 50% Dulbecco's Modified Eagle Medium +50% Ham's F12 Medium containing 5% FBS, 2 mM glutamine, 50 ng/ml Penicillin/Streptomycin (all from Lonza, Switzerland), Choleratoxin (Sigma‐Aldrich, MO), 10 μg/ml Insulin (Roche, Switzerland), 100 μg/ml Hydrocortisone (Sigma‐Aldrich), and 20 ng/ml EGF (PeproTech, NJ) at 37°C in 5% CO2. Cells were grown on glass coverslips, coated with gelatin 0.5% (wt/vol) in PBS, and, at 70% confluence, fixed for 10 min in 4% paraformaldehyde (wt/vol). To detect actively DNA‐replicating cells, Ethinyl‐deoxyuridine (EdU) (Life Technologies, CA) was added to the culture media (final concentration 10 μM) 40 min before fixation. After labeling, cells were washed twice with PBS and fixed as previously described.
Edu Staining and Immunofluorescence of MCF10A cells
For the 7‐parameter widefield microscopy analysis, cells (including the cell lines shown in the Supporting Information Data Section) were permeabilized and processed using the Click‐iT™ EdU Imaging kit (Life‐Technologies) plus Pacific‐Blue azide according to the manufacturer instructions.
After blocking (5% BSA in PBS), cells were incubated for 1 h at room temperature (RT) with primary antibodies [rabbit anti‐53BP1 (ab36823 Abcam, UK) and mouse anti‐p21 (M7202, Dako, Denmark)]. After washing (3×), they were incubated for 1 h at RT with anti‐rabbit Pacific‐Orange‐conjugated IgGs (Life‐Technologies) and anti‐mouse Cy3‐conjugated IgGs (Jackson Immuno‐Research, UK). Cells were then refixed in 4% paraformaldehyde, blocked with 5% BSA containing mouse‐IgG (Jackson Immuno‐Research) and incubated for 1 h with the following mAbs: anti‐KI67 Alexa647‐conjugated (558615, BD Pharmingen, CA), anti‐γH2AX Alexa488‐conjugated (613406, Biolegend, CA), and anti‐human p53 biotin‐conjugated (DO1, Sigma‐Aldrich). Finally, after washings, coverslips were incubated 1 h with CW800‐conjugated anti‐Biotin (600‐132‐098, Rockland, PA). DNA was counterstained with DAPI. Slides were then mounted in Mowiol‐containing mounting media.
For the analysis of p63 expression, a putative stem‐cell marker, the above‐described protocol was modified using an anti‐p63 mAb (sc‐8431, Santa‐Cruz, Germany) and anti‐p21 biotin‐conjugated antibody (ab79467, Abcam). Full paper
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