Ki67 pe

The following antibodies for Western blot, confocal, and ChIP were from Cell Signaling: anti-SNAIL (#3879S), anti-ZEB1 (#D80D3), anti-E-cadherin (#24E10), anti-Vimentin (#D21H3) XP^®. For others: Anti-β-Actin−Peroxidase (A3854; Sigma-Aldrich-Merck), Alexa 488-conjugated secondary antibody (1/400, Molecular Probes), Actin-Stain 488 Phalloidin (1/400; Cytoskeleton, Inc.). FACS and ImageStream antibodies were as follows: CD73-PE antibody (344004; 1:100, Biolegend), Alexa 633-conjugated secondary antibody (1/500; Molecular Probes), Ki67-PE (151210; 1:100; Biolegend).

To determine the cell surface marker expression, cells were harvested with trypsin for flow cytometry analysis. After being blocked with 2.5% FBS, a total of 5 × 10⁵ cells were re-suspended in 200 μl PBS for each reaction. Cells were incubated on ice for 30 min with the following anti-human antibodies, which were conjugated with fluorescein phycoerythrin (PE): CD133-PE, Ki67-PE and CXCR4-PE (BioLegend, USA). Rabbit IgG1-PE (Beyotime, China) was used as the isotype control. Cells were analyzed by flow cytometry (Beckman Coulter, USA).
To determine the intracellular marker expression, cells were harvested and then fixed in a 0.5 ml/tube Fixation Buffer in the dark for 20 min at RT, followed by a 1x wash with Cell Staining Buffer. Cells were then re-suspended in Cell Staining Buffer and stored in 90% FCS/10% DMSO at − 80 °C. The fixed/permeabilized cells were re-suspended in residual Intracellular Staining Perm Wash Buffer and were added a predetermined optimum concentration of PE-conjugated antibody of Ki67 (BioLegend, USA) or rat IgG2b-PE isotype (BioLegend, USA) for 20 min in the dark at RT. Cells were analyzed by flow cytometry (Beckman Coulter, USA).

Single-cell suspensions of lungs isolated from FVB/n;Col1α1-YFP or PyMT;Col1α1-YFP mice were stained using the following antibodies: anti-EpCAM-APC (eBioscience, 17-5791), anti-CD45-PerCP-Cy5.5 (eBioscience, 45-0451), and anti-CD31-PE-Cy7 (eBioscience, 25-0311). DAPI was used to exclude dead cells (Molecular Probes; D3571). Ki67-PE (BioLegend, 652403) intracellular staining of fibroblasts was done using an intracellular staining kit (BD Biosciences, 554714) according to the manufacturer’s protocol. Flow cytometric analysis was performed using CytoFLEX Flow Cytometer (Beckman Coulter). Cell sorting was performed using BD FACSAria II or BD FACSAria Fusion (BD Biosciences). Data analysis was performed with the Kaluza Flow Analysis software (Beckman Coulter).

Flow cytometry (FC) was conducted using the MACSQuant® Analyzer 10 (Miltenyi Biotec) and the following antibodies: CD34-FITC (Stemcell), ki67-PE, Annexin V-APC, and 7-AAD (all bought from Biolegend).

PI staining was used for the cell cycle analysis. Briefly, cells were harvested, suspended in phosphate-buffered saline (PBS) and fixed with 70% ethanol on ice for 1 h. Then cells were washed with PBS and RNA was digested with RNase. Haploid and diploid DNA were labeled with PI overnight. Ki-67/PI double staining was also employed to detect cells in G0 phase. Briefly, suspended cells were treated with Cytofix/Cytoperm buffer (BD Pharmingen) and stained with Ki-67-PE (Biolegend, San Diego, CA) and Hoechst33342 (Sigma). An LSRII flow cytometer was used for the cell cycle analysis according to standard protocols. Experiments were repeated at least three times.