Cd29 fitc

Single-cell suspension of isolated primary mouse mammary epithelial cells (MMECs) was resuspended in 0.2% BSA in Dulbecco's PBS, and the cells were incubated with the following primary antibodies for analysis: CD29-FITC (Miltenyi Biotech, 130-102-975); CD24-APC (M1/69, BioLegend, 101814); CD31-BV421 (MEC13.3, BD Biosciences, 562939); CD45-BV421 (30 F11, BD Biosciences, 563890); Ter119-BV421 (BD Biosciences, 563998); Sca1-BV711 (D7, BioLegend, 108131); and CD49b-PE (HMα2, BioLegend, 103506). All antibodies were diluted 1:500 at 4°C for 30 min. Cells were washed with PBS and resuspended in 0.2% BSA in Dulbecco's PBS with 1:500 Sytox Blue (Life Technologies, Thermo Fisher Scientific) to exclude dead cells. Sorting was performed using a FACSAria Fusion cell sorter (Becton Dickinson). FlowJo V10 was used for post-analysis of sorted cells.

Freshly isolated cells were characterized for ADSVCs surface protein expression [17 (link)] by flow cytometry (MACSQuant analyzer, Miltenyi-Biotech) according to the manufacturer’s instructions. Cells were stained with the following antihuman-conjugated monoclonal antibodies: CD13-APC, CD14-PE, CD29-FITC, CD31-APC, CD34-PE, CD45-VioBlue, CD73-APC, CD90-FITC, CD105-VioBlue, CD144 (VE-Cadherin)-PE, CD146-Biotin, CD166-Biotin, HLA-ABC-FITC, HLA-DR-VioBlue, or relevant isotype-matched controls (Miltenyi-Biotech). Isotypes controls and automated compensation were settled to minimize false positive fluorescence and spectral overlap of fluorochromes respectively. Cell viability and apoptosis were assessed by the 7AAD/AnnexinV/PI assay. In fact, cell viability was first assessed manually using the Trypan blue (Sigma-Aldrich) exclusion assay, and the results were then validated by the 7AAD method by flow cytometry. The telomerase activity [18 (link)] was assessed by real-time qPCR (LightCycler 2.0, Roche, Basel, Switzerland) using the Quantitative Telomerase Detection Kit (Cat#MT3012, Allied Biotech, Taipei, Taiwan) according to the manufacturer’s instructions.