Liberase ci purified enzyme blend

Ear tissue was prepared as previously described.^{40 (link)} Briefly, the two sheets of infected ear dermis were separated, deposited in DMEM containing 100 U/ml penicillin, 100 μg/ml streptomycin and 0.2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp., Chicago, IL, USA) and incubated for 1 h and 30 min at 37 °C. Digested tissue was placed in a grinder and processed in a tissue homogenizer (Medimachine; Becton Dickenson, Chicago, IL, USA). To obtain neutrophils recruited to the site of infection in the skin, LYS-eGFP mice were inoculated in the ear dermis with 1–2 × 10⁶Lm-RFP or Lm-NT-OVA-RFP or 1 × 10⁶T. gondii-RFP parasites in 10 μl. After 12 h, ear tissue was prepared as described above and infected (eGFP^hiRFP⁺) and uninfected (eGFP^hiRFP⁻) neutrophils were sorted from dermal tissue using a FACSVantage or a FACsAria (BD Biosciences, Chicago, IL, USA) cell sorter. Sorted populations were washed once and immediately analyzed for apoptotic markers or cultured with mouse BM-DCs. Presorted neutrophil populations recruited to the site at 2 and 16 h after infection were stained with Annexin-V-APC and 7-AAD (BD Biosciences), and cells were analyzed by flow cytometry.

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”.

C57BL/6 mice ears were intradermally injected with LuloHya (10 μg and 1 μg), Lundep (10 μg and 1 μg), Lu. longipalpis SGE (equivalent to 2 pairs of salivary glands). As negative controls, ears were injected either with PBS or with a non-related salivary protein from the mosquito Ae. aegypti which was expressed and purified in the same manner. After 2 h, mice were euthanized, and the two sheets of ear dermis were separated, deposited in PBS containing 0.2 mg/ml Liberase CI purified enzyme blend (Roche Diagnostics Corp.), and incubated for 1 h at 37°C. Digested tissue was placed in a grinder and processed in a tissue homogenizer (Medimachine; Becton Dickenson). Tissue homogenates were filtered using a 30 μm Filcon filters (BD). The resulting single cell suspensions were first stained with the Fixable Yellow Dead Cell Stain Kit (Invitrogen) for 20 min. The suspension was then washed and incubated with anti-Fc (CD16/32) antibodies to block non-specific binding. After 10 min, the cells were stained for Ly6C (clone AL-21; FITC; BD), Ly6G (clone 1A8; PE; BD) and CD11b (clone M1/70; PE-Cy7; BD) for 30 min. Cells were gated based on forward scatter and side scatter parameters and further gated on live cells. Cells were acquired on a MACSQuant flow cytometer (Miltenyi Biotec) and data were analyzed with FlowJo Software 4.3.