Sca 1 pe cy7

Progenitors in the peripheral blood were identified and quantified using a protocol adapted from Serwold et al. 40 (link). Peripheral blood from six animals per group was collected in EDTA by terminal cardiac bleed and pooled. Blood was diluted 1:1 in RPMI and mononuclear cells enriched by centrifuging through a layer of Lympholyte-Mammal (Cedarlane Laboratories, Burlington, USA). Cells were collected, washed, and stained for Lin markers (hematopoietic lineage eFluor450 cocktail (EBioscience) and NK1.1, CD11b, and CD19 on FITC), CD27-PerCP-eFluor710, CD127-Alexa Fluor 647, Sca-1-Pe-Cy7, CD117-allophycocyanin-eFluor780, and Flt3-PE (all eBioscience). At least 5 × 10⁶ events were analyzed by flow cytometry per sample.

The following antibodies were obtained from BD Biosciences (San Diego, CA, USA): anti-CD3-APC (145-2C11), anti-cKit-Pe-Cy7 (2B8), and anti CD11b-PE (M1/70). For Lineage depletion these markers were used: CD3 (145-2C11), CD4 (L3T4), CD8 (53-6.7), CD11b (M1/70), Gr1 (RB6-8C5), B220 (Ra3-6B2), Ter119 (Ly76) and Nk1.1 (PK136) biotin and subsequently were stained with streptavidin eFluor 450 (48-4317) from eBioscience (Vienna, Austria). The following antibodies were also purchased from eBioscience: Ly5.1-PE-Cy7 (A20), Ly5.2 Alexa Fluor 780 (104), B220 PE-Cy7 (RA3-6B2), Gr1 eFluor 450 (RB6-8C5) and Sca1 PE-Cy7 (D7). Cells were stained in fluorescence activated cell sorter (FACS) buffer (PBS, 2% bovine serum albumin, 0.1% sodium azide) for 30 min at 4 °C. Ultimately, cells were washed and measured either on a Canto I, or an Aria (BD Biosciences) FACS. For FL LSK, Mac1 was precluded from the lineage gate, as FL LSK express Mac1.^{45 (link)} For apoptosis analysis, E14 FL cells were stained with 7AAD/AnnexinV kit (BD Bioscience) in combination with LSK staining. For proliferation analysis E14 FL cells were stained with PE mouse anti-Ki67 set (BD Pharmingen, San Diego, CA, USA) in combination with LSK staining or for cell cycle analysis with an adapted protocol for combined LSK and propidium iodide staining.^{46 (link)} Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

For isolation of anagen cells, skins were first incubated in 0.25% collagenase (Sigma) in HBSS (GIBCO) at 37°C for 1–2 hr to digest the dermis. Next, the digested tissues were centrifuged and resuspended in 0.25% Trypsin (GIBCO) and incubated at 37°C for 20 min with gentle shaking. Single-cell suspensions were obtained by pipetting gently and filtering through 70 μm strainers, followed by 40 μm strainers. Staining buffer (PBS with 5% FBS treated with Bio-Rad Chelex to remove calcium) was added to inactivate Trypsin, and cells were collected by centrifugation for 5 min at 300 × g. Cell suspensions were incubated with the appropriate antibodies diluted in staining buffer for 15 min at 4°C. For the isolation of telogen cells, subcutaneous fat was removed from skins with a scalpel. Skins were placed on 0.25% Trypsin at 37°C for 30 min with the dermis side facing down. Skins were then gently scalped from the epidermis to isolate epidermal cells and filtered, centrifuged, and stained as above. The following antibodies were used: CD34–Alexa 660 (eBioscience), α6–phycoerythrin (eBioscience), Sca1–PE-Cy7 (eBioscience), and c-Kit-APC (eBioscience).