Itga6 pe cy7

Cell proliferation was assessed using the Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Cells were grown for 9–14 days in 3D–3C culture before 10 µM EdU was added to the medium and incubated for 2 h or 24 h. Matrigel droplets were degraded, the cells were washed with PBS and subsequently stained with fixable LIVE/DEAD-violet dye (Thermo Fisher Scientific, 1:500) for 20 min at RT. The cells were washed with FACS buffer and surface marker staining was performed for 30 min on ice using CD34-eFluor 660 (eBioscience, clone RAM34, 1:100) and ITGA6-PE/Cy7 (Biolegend, clone GoH3, 1:1000). The cells were washed with 1% bovine serum albumin (BSA) in PBS, fixed with 4% PFA for 10 min at RT and permeabilized using Click-iT saponin-based permeabilization buffer for 15 min at RT. The EdU reaction cocktail was prepared following the manufacturer's instructions and incubated for 30 min at RT in the dark after washing the cells. The cells were analyzed on FACSCantoII.

Matrigel droplets were degraded in 0.5% trypsin, 0.5 mM EDTA in PBS for 8 min at 37°C. Trypsin was neutralized using cold KGM. After centrifugation for 5 min at 600 g, cells were washed with FACS buffer (2% FBS, 2 mM EDTA in PBS). Surface marker staining was performed for 30 min on ice with the following antibodies: CD34-eFluor 660 (eBioscience, clone RAM34, 1:100) and ITGA6-PE/Cy7 (Biolegend, clone GoH3, 1:1000) for sorting or CD34-eFluor 660 (eBioscience, clone RAM34, 1:100) and ITGA6-Pacific Blue (Biolegend, clone GoH3, 1:200) for analysis. Freshly isolated keratinocytes were stained additionally with Sca1-Pacific Blue (Biolegend, clone D7, 1:400). 7AAD or propidium iodide (PI) was used to exclude dead cells. Cells were analyzed on FACSCantoII (BD Biosciences) or sorted on FACSAria IIIu Svea and FACSAria Fusion sorters (BD Biosciences). Sorted cells were collected in 15 ml tubes containing KGM at 4°C.