Dispase 2

Dorsal skin was obtained from newborn mice and immersed in Dispase II solution, 2.5 U/ml Dispase II (Roche, Indianapolis, IN) with antibiotic-antimycotic solution CnT-ABM10 (CELLnTEC, Bern, Switzerland) in CnT-PR medium (CELLnTEC) for 16 h at 4°C. After being washed with PBS, the sheet of epidermis, separated from the dermis, was floated on a 500 μl drop of TrypLE select (Thermo Fisher Scientific) for 25 min at RT. After addition of 2 ml of medium to the drop, the sheet of epidermis was shaken gently on the drop to disperse the keratinocytes. The cell suspension was collected in a 15-ml tube containing fetal bovine serum (FBS). The cells were filtered through a 70-μm cell strainer to remove clumps and debris, centrifuged for 7 min at 200 × g, suspended in medium with 10% FBS and centrifuged again for 7 min at 1300 rpm. The cells were plated in CnT-PR medium with 10% FBS at 1.5 × 10⁵ cells/cm² and incubated at 37°C in a humidified 5% CO₂ incubator. The medium was removed 16 h after plating and replaced with CnT-PR medium without FBS. The medium was changed every other day.

For cell isolation from lung tissues, mice were euthanised (600 mg/kg pentobarbital/17 mg/kg mepivacaine) and then perfused with 10 ml of ice-cold PBS through the right ventricle of the heart. 1.5 ml Dispase II (Roche) (5 mg/ml in IMDM) was then injected intratracheally into the lungs, followed by 0.4 ml 1% low-gelling agarose solution (in PBS) (Sigma). Mice were then placed on ice allowing the agarose/dispase-filled lungs to set. Lungs were then dissected and placed in 2 ml Dispase II solution for 30 min to dissociate epithelial cells. Lungs were passed through a 100 μm strainer, before a 10-min DNase I digestion (50 μg/ml) (Sigma). Following digestion, lung homogenates were passed through a 70 μm strainer and centrifuged at 1400 r.p.m. for 5 min at 4 °C, before red blood cell lysis. Single-cell suspensions were preincubated with anti-FcgRIII/II (Fc block), before a 30-min incubation with the indicated fluorochrome-labelled antibodies. For Ki67 staining, cells were fixed and permeabilised before staining. Cells were analysed using a Fortessa X20 (Becton Dickinson).
Airway epithelial cells from ex vivo tracheae were dissociated using Dispase II (Roche) (5 mg/ml in IMDM), for 2 h at 37 °C. After incubation, dissociated tissues were washed in media containing 10% FCS, passed through a 70 μm strainer, pelleted and stained with the indicated antibody panel for flow cytometry.

Epithelial crypts (ECs) and lamina propria cells were isolated from colon as previously reported (Wang et al., 2018 (link)). Briefly, ECs were collected using HBSS buffer supplemented with 2% FBS, 5 mM EDTA and 1 mM DTT (American Bioanalytical). Single epithelial cell suspensions were made by digestion of crypts in HBSS containing 0.5 mg/ml of dispase II (Roche) at 37°C for 10 min with intermittent shaking. Lamina propria leukocytes (LPLs) were isolated by digestion of lamina propria tissues in Dulbecco's PBS with 10% FBS, 0.5 mg/ml dispase II, 0.5 mg/ml collagenase D (Roche), and 100 U DNase I (Sigma) at 37°C for two consecutive 20 min. LPLs were then recovered by Percoll gradient centrifugation at 1,000 g for 20 min.