Dispase 1

BMDM were differentiated from bone marrow incubated in DMEM with 10% fetal calf serum (FCS), 2 mM L-glutamine, 10U/ml Penicillin, 10 μg/ml Streptomycin, and 10 ng/ml M-CSF (GenScript) for 7 days.
NIH 3T3 cells (mouse, male, ATCC CRL-1658) were maintained in DMEM supplemented with 10% Newborn Calf Serum (NBCS), 2 mM L-glutamine, 10U/ml Penicillin, and 10 μg/ml Streptomycin.
Primary corneal epithelial cells were collected from C57Bl/6 mice by incubating eyes overnight at 4 °C in a 1:1 mixture of DMEM:F12 supplemented with 4 mg/ml Dispase I (Sigma–Aldrich), 10U/ml Penicillin, 10 μg/ml Streptomycin, and 0.025 μg/ml Amphotericin B. Epithelial sheets were peeled from eyes and dissociated in TrypLE for 10 min at 37 °C with gentle agitation. Cells were passed through a 0.45 μm strainer (Corning), washed, and cultured on collagen and fibronectin-coated plates in KSFM supplemented with 5 ng/ml hEGF, 50 μg/ml Bovine Pituitary Extract, and 100 ng/ml Cholera Toxin.
All cells were maintained at 37 °C with 5% CO₂. Adherent cells were detached for subculture and experiments using TrypLE. Unless otherwise noted, all cell culture materials were purchased from Gibco/Thermo Fisher.

The sample preparation and analysis for RNA sequencing have been described in detail (Gnedeva and Hudspeth, 2015 (link)). For qPCR, utricles at each developmental stage were isolated by microdissections and treated with 0.5% Dispase I (Sigma) for 15 min at 37˚C to isolate the sensory epithelia. For each sample, total RNA from 7 to 12 utricular maculae was isolated by a standard protocol (RNeasy Micro Kit, Qiagen) and used to create a cDNA library. The qPCR primers were designed with PrimerQuest (Integrated DNA Technologies). Relative gene-expression levels were obtained by normalization to the expression of Gapdh in each sample. qPCR analyses were performed on an Applied Biosystems 7900HT Sequence Detection System with FastStart Universal SYBR Green Master mix (Roche Applied Science).

Confluent Monolayers of CHO β₁ cells seeded on 24 well plates were depleted of serum for 24 h, and then incubated with or without ouabain (50 nM) for 24 h. Then the monolayers were washed with ice-cold PBS, and detached from the plates by incubation with PBS without Ca²⁺ supplemented with 0.6 U/mL of Dispase I (D4818, Sigma-Aldrich) for 35 min at 37 °C. Subsequently the Dispase solution was carefully removed using a 200 µL pipette tip, and replaced by 100 μL of PBS. The cells were then mechanically stressed by pipetting up and down 5 times using a 200 µL pipette. The resulting aggregates were visualized by light microscopy using the 10 × and 20 × objectives (Axiovert 200M Fluorescence/Live Cell Imaging, Carl Zeiss, Oberkochen, Germany). Three independent biological replicates were imaged using the AxioVision 4.8 software. The number of aggregates was counted using the Fiji 1.0 software cell counter (aggregates < 200 μm² were excluded from the quantification) [84 (link)].

Ketamine (80 mg/kg per mice) and Xylazine (5 mg/kg per mice) were injected i.p. before tissue preparation. After anesthesia, the skin samples with different phases of hairs were obtained from the back of mice after anesthesia, and the wound was sewed afterwards. Then the skin samples were incubated in 0.25% solution of Dispase (Dispase I, Sigma-Aldrich Co. LLC) in Hanks’ balanced salt solution (HBSS, Life technologies, Thermo fisher Scientific Inc.,Grand Island, NY) at 4 °C overnight. Based on previous research, hair follicles represent grey or black in anagen phase, while showing pink with no pigment during telogen phase (Maksim & Plikus, 2009 ). And the epidermis with telogen hair follicles or anagen hair follicles was separated with forceps under a binocular light microscope according to skin color and morphology.

Spleens were harvested and perfused with RPMI 1640 medium containing 2% FCS, 20 mM Hepes (all from Lonza), 0.2 mg/ml Collagenase P (Sigma Aldrich), 0.8 U/ml Dispase I (Sigma Aldrich) and 100 µg/ml DNaseI (Applichem) with 22 G syringe. Samples were torn into smaller pieces and incubated at 37 °C for 30 min, with resuspension and collection of supernatant every 15 min to PBS containing 1% FCS and 10 mM EDTA (MACS buffer). To enrich fibroblastic stromal cells, hematopoietic and erythrocytes were depleted by incubating the cell suspension with MACS anti-CD45 and anti-TER119 microbeads (Miltenyi Biotec) and passing them through a MACS LS column (Miltenyi Biotec). Unbound single-cell suspensions were used for further flow cytometric analysis.

For the flow cytometric analyses, organ-specific samples were obtained by pooling the spleens, livers, aortic vessels and gall bladders of the different chicks in Hanks’s buffered saline solution (HBSS) without Ca²⁺ and Mg²⁺ in the dark at 4°C in order to be able to analyse at least 200 000 cells for each organ. Independent infections were repeated at least three times. Gall bladders and aortic vessels were cut into small pieces and samples put in collagenase A (0.3%—Sigma)—dispase I (1 U ml⁻¹—Sigma)—HBSS for 30 min at 37°C. The whole purification process was performed at 4°C. All organs were then homogenized in HBSS using syringe plungers and filtered through 40 µm-mesh cell strainers (BD Falcon, Tewksbury, USA), before being transferred into a 50 ml centrifuge. After centrifugation at 1000 g for 15 min, cells were washed, resuspended in HBSS at approximatively 5.10⁶–1.10⁷ cells ml⁻¹ and maintained in the dark at 4°C.