Bacillus licheniformis protease

Cold active protease (CAP), was prepared and stored on ice (5 mM CaCl2, Sigma cat#21115, 10 mg/mL Bacillus Licheniformis protease, Sigma cat#P5459, 12.5 U/mL DNAse, Sigma cat#4716728001). Mice were perfused with 20 mL of CAP using a small vein infusion set (Kawasumi cat#D3K2-23G) and two 10 mL syringes per mouse. Bladders were dissected and immediately put in a 60 mm × 15 mm petri dish (Fisherbrand cat#FB0875713A) containing CAP on ice for 10 min. The bladders were then transferred to HBSS media (ThermoFisher cat#14170-112) containing 1% bovine serum albumin (Sigma cat#A2058) and 0.1% glucose, where they were inverted and the urothelium was manually separated from the stroma. The cell suspension was then collected into a 1.5 mL Eppendorf tube on ice and gently triturated until the cells were in a single-cell suspension.

A psychrophilic protease protocol for single cell dissociation was modified from previous report (Adam et al., 2017 (link)). Mandibles from E10.5 wildtype CD1 mouse embryos were rapidly dissected in ice-cold PBS. Five embryonic mandibles were pooled in a sterile 1.5 ml microcentrifuge tube with 350 µl of freshly made protease solution containing 5 mg/ml of Bacillus Licheniformis protease (Sigma P5380) in DPBS, 5 mM CaCl2, and 125 U/ml DNase (Applichem A3778). The sample was incubated in 4°C for a total 8 min, with trituration for 15 s every 2 min using a 1 ml pipet. Single cell dissociation was confirmed by observation of samples under microscope. Following inactivation of the protease with ice-cold DMEM containing 10% FBS, the cells were pelleted by centrifugation at 1200 g for 5 min. The cells were resuspended in 1 ml ice-cold DMEM containing 10% FBS, and filtered through a sterile 40 μM filter (BD Falcon 352340) and pelleted by centrifugation at 1200 g for 5 min. The dissociated cells were washed with ice-cold DMEM containing 10% FBS. Cell number and viability were analyzed using a hemocytometer following trypan blue (Gibco 15250061) staining. The single cell preparation displayed greater than 90% viability in this experiment.