Filcon filter

After exposure to sand fly bites, mice ears were harvested and disinfected with ethanol. The dermal sheets were then separated using forceps and digested in PBS containing Liberase TL (Roche) at 37°C for 1 h. The digested tissue was further homogenized by mechanical disaggregation in a BD^TM Medimachine (BD Biosciences, Franklin Lakes, NJ, USA) and filtered in a 50 μm Filcon filter (BD Biosciences, Franklin Lakes, NJ, USA). Single cell suspensions were treated with anti-Fc (CD16/32) antibodies to block non-specific binding. After 15 min, cells were stained for Ly6C (clone AL-21; FITC; Biolegend), Ly6G (clone 1A8; APC-Cy7; Biolegend), CD11b (clone M1/70; PE-Cy7; Biolegend), F4/80 (clone BM8; PerCP; Biolegend) at a concentration of 1:100, and with LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit (1:1,000, Thermo Fisher Scientific, Waltham, MA, USA). Stained samples were sorted on a FACSAria-FUSION cell sorter using DIVA 6.1.8 software (BD Biosciences, San Jose, CA). Cell subsets were sorted at 4°C in chilled collection tubes supplemented with PMSF (Sigma-Aldrich, St. Louis, MO, USA). Collected cell subsets were pelleted down and lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min on ice. Immunoblotting against HO-1 (ab13248, abcam, Cambridge, MA, USA) and Histones H3 (#4499, Cell Signaling, Danvers, MA, USA) was carried out as previously described.

80–100 ovary pairs per genotype were hand-dissected in Schneider's media with 5% fetal calf serum on ice. The dissected ovaries were rinsed three times in chilled PBS and then incubated for 10 min at room temperature in a dissociation solution comprising of 2 mg/ml Collagenase in 0.5% Trypsin solution in PBS with intermittent vigorous shaking. After settlement of debris, the suspension was filtered through a 40 µm Filcon^® filter (BD Biosciences) followed by centrifugation at 500×g for 5 min at 4°C. The resulting pellet was suspended in 1 ml of serum-free Schneider's medium containing 10 µg/ml propidium iodide (PI). After 30 min incubation at room temperature, samples were sorted on a BD FACSARIA™ II cell sorter using GFP^high and PI^low fluorescence intensity channels. Sample gating and acquisition were performed using BD FACSDIVA™ software (version 6.1.2).

Single cell dermal isolations were generated from P1.5 or 8-week-old mice and sorted by FACS to obtain melanoblasts or McSCs, respectively. Cells of the melanocyte lineage were identified by their surface expression of the receptor KIT. Both melanoblasts and McSCs reside in the hair follicle at these respective time points and exhibit KIT+ immunolabeling (Figs 2 and 3). This isolation method was adapted from previously published protocols [83 (link)][84 ]. In brief, trunk skin was incubated in 0.25% Trypsin-EDTA to separate the dermis from the epidermis. The dermis was further dissociated by enzymatic treatment with 0.3 mg/mL Liberase TL (Roche) and then physically disrupted by passing the dermal cell solution forcefully through a syringe-type, 70 uM Filcon filter (BD Biosciences). Dermal cells were labeled with the cell surface markers, KIT (CD117, BD Pharmingen) and CD45.2 (BD Pharmingen), and assessed for fluorescence on a FACSAria (Becton Dickinson). Cells of the melanocyte lineage were positively selected for by gating on the KIT+, CD45.2− cell population. Mast cells were negatively selected away by removing any double positive cells (KIT+, CD45.2+) (Fig 2). Analysis of the cell populations within FACS gates was performed using FlowJo software.