Ab119695

The mammary colony forming cell assay is used to assess the differentiation function of luminal progenitor cells. It is known that luminal progenitor cells can form distinct colonies when cultured on plates pre-seeded with irradiated NIT-3T3 fibroblasts [23 (link), 35 (link)]. In this study, the colony forming cell assay was performed [13 (link)]. However, instead of Giemsa staining, colonies were stained with basal and luminal markers of keratin 14 (K14) and K8, respectively, using immunofluorescence (IF). In detail, colonies were fixed with 100% cold methanol for 1 min, washed with phosphate buffer solution with 0.05% Tween 20 (PBST), blocked with 10% serum/1%BSA/0.3M glycine in 0.1% PBS-Tween 20 for 30 min, and incubated with primary antibodies of K14 (rabbit monoclonal IgG, ab119695, 1:200 dilution, Abcam) and K8 (rabbit monoclonal IgG, ab53280, 1:200 dilution, Abcam) diluted in 5% serum/1%BSA/0.3M glycine in 0.1% PBS-Tween 20 at 4°C overnight. Colonies were then washed with PBST twice before being incubated with secondary antibodies diluted in 1%BSA/0.3M glycine in 0.1% PBS-Tween 20 at room temperature for 1 h. Finally, colonies were washed with PBST and stained with DAPI for fluorescent imaging. A minimum of 20 colonies per animal (× 3 animals) per treatment group were assessed with IF.

The human hair follicloids were first blocked in PBS containing 3% goat serum (Abcam, Cambridge, CB2 0AX, UK) and 0.3% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA) for 1 h at 25 °C and subsequently incubated overnight with anti-cytokeratin 14 SP53 (ab119695; Abcam), anti-K40 AE13 (ab16113; Abcam), and anti-versican (ab19345, Abcam) antibodies at 4 °C. The samples were incubated with the corresponding secondary antibodies (Alexa Fluor® 488 ab150077; Abcam) suspended in the blocking solution for 2 h at 25 °C and finally stained with 4′,6-diamidino-2-phenylindole in PBS for 10 min. Next, 10-μm-thick frozen sections were prepared using the same steps as histological staining. An all-in-one fluorescence microscope (BZ-X810, Keyence) was used for fluorescence imaging.

EpSCs were isolated from the backs of newborn (0–3 days) SD rat skin. Briefly, skin samples were taken from the backs of SD rats and cut into pieces (0.2×0.3 cm). After incubation in 0.1% Dispase II in PBS at 4°C overnight, the epidermal sheet was carefully separated from the dermis, minced, and then digested in 0.25% trypsin in PBS for 10 min at 37°C. Trypsin was inactivated in calcium-free RPMI 1640 medium containing 10% FBS. Following filtering and centrifugation, the cells were resuspended in keratinocyte serum-free medium (KM, #2101; ScienCell) and seeded at a density of 10⁵ cells/cm² in flasks coated with 100 μg/ml collagen IV (C5533; Sigma) to adhere for 20 min at 37°C. The nonadherent cells were subsequently discarded, and rapidly adhering cells were cultured in KM at 37°C in 5% CO₂. Meanwhile, the cells were identified as EpSCs with CK19^bri (10712-1-AP; Proteintech), CD34^bri (ab119695; Abcam), and CD71^dim by flow cytometry (data not shown).