Isolation and Enumeration of Murine Splenocytes and Ear Cells

Euthanasia was performed by cervical dislocation always under isoflurane anaesthesia (Piramal healthcare, UK). Spleens were aseptically collected from euthanized animals, weighed, and homogenized using Falcon 100 µm Cell Strainers (Corning Life Sciences, MA, USA). Ears were collected, immersed in 70% ethanol and then in PBS. Ear cell suspensions were obtained as previously described^{43 (link)}. Briefly, the ventral and dorsal sheets were separated, deposited in RPMI containing 0.25 mg/ml Liberase CI purified enzyme blend and 10 µg/ml DNAse (both from Roche, Switzerland), and incubated for 1 h at 37 °C and 5% CO₂. Digested ear sheets were subsequently homogenized for 3 min using the Medicon/Medimachine tissue homogenizer system (BD, NJ, USA) and the resultant single cell suspensions were filtered using a 70 µm-pore size Falcon cell strainer (BD Biosciences, NJ, USA). Viable cell numbers were determined for all obtained cell suspensions (ear and spleen derived) under the microscope, using a Neubauer chamber and trypan blue solution as the “viability marker”.