α6 integrin buv395

Mouse tail skin was incubated in 0.25% Trypsin/EDTA overnight at 4°C and for 30 min at 37°C. Single cell solution was prepared by scraping and subsequent filtering with strainers (70 μm, followed by 40μm). Cells were stained with the following antibodies for 30 min on ice: CD34-biotin (1:50, eBioscience, 13–0341), Streptavidin-APC (1:100, BD Biosciences, 554067), α6-integrin-BUV395 (1:100, BD Biosciences, custom order) and Sca1-BV421 (1:100, BD Biosciences, 562729). The dead cells were excluded by using propidium iodide (P4864, Sigma Aldrich). FACS (Fluorescence-activated cell sorting) analyses were performed with FACS Aria (BD Biosciences). The data was processed with FlowJo software to measure the mean fluorescence intensity of basal markers. For the detection of apoptotic cells, cells were stained with PE Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend) according to the manufacturer’s instructions.

Mouse dorsal and ventral skin were dissected and the subcutaneous and fat tissues were removed from the dermal side of the skin. The skin was incubated in 0.25% trypsin/versene overnight at 4°C and for 30 min at 37°C. The single‐cell solution was prepared by scraping the epidermis and subsequent filtering with strainers (70 μm, followed by 40 μm). Cells were stained with the following antibodies for 30 min on ice: CD34‐biotin (1:50, eBioscience), Streptavidin‐APC (1:100, BD Biosciences), α6‐integrin‐BUV395 (1:100, BD Biosciences, custom order) and Sca1‐BV421 (1:100, BD Biosciences). The dead cells were excluded by staining with propidium iodide (Sigma‐Aldrich). Cell isolation was performed with FACS Aria (BD Biosciences), and the data were analyzed with the FlowJo software (BD, Franklin Lakes, NJ).