In cell 2200 automated 991 microscope

Cells grown in 96-well plates were washed with PBS and fixed in 4% paraformaldehyde for 15 min at RT. Cell were then washed in PBS twice before being permeabilized and blocked for 40 min with 0.2% Triton X-100 together with 1% bovine serum albumin and 0.2% gelatin fish (Sigma). For immunofluorescence (IF) staining, cells were incubated overnight with the primary antibody (Supplementary Fig. S5); in the case of BrdU, cells were treated with DNaseI and MgCl₂. Cells were then washed in PBS and incubated 1 h with secondary antibody, DAPI (Sigma-Aldrich). IF images were acquired using IN Cell 2200 automated 991 microscope (GE) and the IN Cell 2200 Developer software version 1.8 (GE).

Cells were grown in a 96-well plate and fixed with 4% paraformaldehyde for 10 min at RT. Cells were then washed twice with PBS and permeabilized by incubating with 0.4% Triton X-100 in PBS for 10 min at RT. After a PBS wash, cells were blocked 30 min at RT using 1% BSA in PBS supplemented with 0.1% Tween-20 (PBST). Primary antibodies (details found at the end of the section) were diluted in 1% BSA-PBST and incubated overnight. For BrdU staining, cells were treated with DNaseI and MgCl₂ simultaneously with the primary antibody. Cells were then washed with PBS and incubated with their respective secondary antibody for 1 h at RT. Nuclei were stained with DAPI (Sigma-Aldrich). Images were acquired using INCell 2200 automated 991 microscope (GE) and INCell 2200 Developer software version 1.8 (GE) was used for image analysis. Antibodies used in this study are: p21^CIP (Abcam, Cat# ab109520), Ki67 (Abcam, Cat# ab92742), BrdU (Abcam, ab6326; 1:500) and F9 (Proteintech, Cat# 21481–1-AP).