Cd24 percpcy5

Dermal fibroblasts were isolated as previously described (Jensen et al., 2010 (link); Collins et al., 2011 (link); Rognoni et al., 2018 (link)). The muscle and fat from dissected neonatal or adult back skin were scrapped off from the underside of the skin using a scalpel and the tissue was incubated overnight at 4∘C in a Dispase (Sigma) only solution. The epidermis was then carefully peeled off and discarded, leaving the intact dermis. The dermis was minced using a scalpel and enzymatically dissociated with a mixture of 1.25 mg/ml collagenase type 1 (Invitrogen), 0.5 mg/ml collagenase type 2 Worthington), 0.5 mg/ml collagenase type 1V (Sigma), 0.1 mg/ml hyaluronidase IVS (Sigma) and 50 U/ml DNase 1 for approximately 45 min at 37∘C. Dermal cell suspensions were passed through a 70 μm cell strainer and washed three times with PBS before being labeled with the following antibodies: Cd24 PerCP-Cy5.5 (eBioscience, Clone M1/69), Cd26 PerCP-Cy5.5 (eBioscience, Clone M194-112), Ly-6A/E APC (eBioscience, Clone D7), Cd140b APC (Thermo Fisher Scientific, 17-1402-82), Pref-1/Dlk1 APC (R&D Systems, FAB8634A), Lrig1 Alexa Fluor 488-conjugated (R&D Systems, FAB3688G). DAPI was used to exclude dead cells. FACS analysis was carried out with BD FACSCanto 1 or 11. Data analysis was performed using FlowJo software version 10.5.3.