Dispase 2

Ovine abomasum epithelial cells were isolated from six 3 cm² gut fold sections collected at post-mortem from two sheep 21-days post T. circumcincta infection, following 10x Genomics single cell protocol. Tissue sections were placed in ice-cold Hanks’ Balanced Salt Solution (HBSS) containing 2.5% fetal bovine serum (FBS), two sections of tissue per tube. Tissue was washed with HBSS, shaking gently to remove any stomach contents, then incubated with HBSS containing 2.5 mM EDTA (Sigma Aldrich), 1mM DTT (Sigma Aldrich) and 10 μg/ml DNase I (Roche) at 37°C for 20 min with agitation. Tubes were shaken vigorously, and the supernatant centrifuged at 330 x g for 5 min to pellet released cells, which were washed in HBSS, centrifuged again and re-suspended in HBSS containing 1 U/ml Dispase II (Stem Cell Technologies, 07913) and 10 μg/ml DNase I (Sigma, 10104159001) and incubated for 10 min at 37°C with agitation. FBS (250 μl) was added to each tube, which was then centrifuged. Following a final wash in HBSS, cells from the same sheep were pooled in 5 ml HBSS and strained through a 70 μm Nylon mesh sterile strainer (Fisherbrand). FBS (250 μl) was added, samples centrifuged and pellets re-suspended in 5 ml PBS. Cell count and viability assessment were performed with Trypan Blue staining.

Samples of the whole trunk between somite 15 and 20 were taken from embryos at 2, 3 and 4 days of development. The explants were incubated in Dispase II (Stem Cell Technologies; #07923, at 1U/mL in DMEM) at 37°C for 20 minutes to degrade collagens and fibronectin. Tissues were then separated using mounted needles. Neural tube explants were then incubated in a trypsin solution (ThermoFisher, 25300054) to generate single cells. From neural tube explants from 3-day and 4-day old embryos, numerous cells (most likely neurons) did not adopt a round morphology after dissociation, instead they maintained an elongated fiber-like morphology and accumulated at the bottom of the tubes. They were not included in the supernatant used for cell diameter analysis.

The small intestine and/or colon were removed and flushed with ice-cold sterile PBS using a 19-gauge feeding needle. The small intestine was further subdivided into the duodenum (proximal 7 cm), jejunum (next 10 cm), and ileum (distal 10 cm), and Peyer patches were removed. Both small intestinal regions and colons were then opened longitudinally and gently agitated at 4°C in PBS, 2% FBS, 5 mM HEPES, and 1 mM DTT for 10 min. The tissue was then transferred into prewarmed PBS, 2% FBS, 5 mM HEPES, and 5 mM EDTA and rotated at 37°C for 15 min followed by vigorous shaking to remove epithelial cells. This was repeated and epithelial cells from both fractions were combined and washed with PBS. The epithelium was then digested in DMEM containing 10% FBS, 0.5 U/ml Dispase II (StemCell Technologies), and 50 μg/ml DNase (Roche) for 10 min at 37°C. The resulting solution was passed through 40-μm filters and washed with PBS, 2% FBS, and 1 mM EDTA. The resulting single-cell suspension was initially Fc blocked with anti-CD16/CD32 (clone 93; BioLegend) and then stained with the following Abs: PacBlue-conjugated anti-CD45 (clone 30-F11; BioLegend) and allophycocyanin-conjugated anti-EpCam (clone G8.8; BioLegend). Cell viability was assessed by propidium iodide (BioLegend) staining.

Distal small intestinal organoids were prepared as previously described (14 (link)). When indicated, IL-13 (10 ng/ml, endotoxin level < 0.01 ng/μg; BioLegend) was added to organoid media for 48 h. To perform flow cytometry, organoids were liberated from the matrigel matrix as described (14 (link)) and digested in DMEM containing 10% FBS, 0.5 U/ml Dispase II (StemCell Technologies), and 50 μg/ml DNase (Roche) for 8 min at 37°C. The resulting solution was filtered through 40-μm mesh and stained for flow cytometry with allophycocyanin-conjugated anti-EpCam (clone G8.8; BioLegend) with cell viability assessed with propidium iodide (BioLegend).

Isolation of DRG neurons was performed according to Rossbach et al. et al. (2011) [2 (link)]. To collect DRGs, mice were deeply anaesthetized with CO₂ and then exsanguinated. Then, 15–20 DRGs were collected along the whole opened vertebral column. DRGs were enzymatically digested in dispase II (2.5 mg/mL; Stemcell Technologies, Vancouver, Canada) and collagenase from Clostridium histolyticum (2.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) dissolved in Ca²⁺- and Mg²⁺-free Hank’s Buffered Salt solution (Thermo Fisher Scientific, Fair Lawn, NJ, USA) for a total of 60 min at 37 °C. Neurons were dissociated every 30 min. with pasteur pipettes.
The cells were washed with DMEM medium (Corning, Manassas, VA, USA) containing 10% FBS (Mediatech Inc., Manassas, VA, USA) and 1% Penicillin/Streptomycin (Pen/Strep; Corning, Manassas, VA, USA) by centrifugation and resuspended in 160 µL media. Then, 20 µL of the cell suspension were transferred to poly-l-lysine hydrobromide (0.1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and laminin (0.1 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) coated glass coverslips (18 mm, round; Warner Instruments; Hamden, CT, USA) and incubated for 2 h at 37 °C and then flooded with a larger volume of DMEM/10% FBS/1% Pen/Strep. Cells were incubated at 37 °C overnight until measurements were performed less than 24 h later.

10-somite long portion of the trunk at the level of the prospective forelimb region were dissected from embryos at stage HH12. Explants were then cultured in suspension in DMEM for 2 hours at 37°C with Rock inhibitor (Y27632), Rac1 inhibitor (NSC 23766) at 400 μM, 1/250 dilution from 100mM stock solution or Dispase II (Stem Cell Technologies; #07923, at 0.2U/mL). All drugs are dissolved in DMSO, therefore control conditions for the drugs correspond to DMSO only at 0.4% in DMEM.