α ly6g apc

Blood cell suspensions were prepared as described above and incubated with the FC blocking antibody α-CD16/32 (eBioscience, San Diego, CA; [5 μg/ml]) for 10 min. The cells were subsequently incubated with the following antibody panel for 25 mins. on ice: α-F4/80-AF488 (AbD serotec, Oxford, UK; [0.5 μg/ml]), α-CD115-PE (Biolegend, San Diego, CA; [1 μg/ml]), α-CD11b-PE/Cy7 (Biolegend; [0.5 μg/ml]), α-Ly6C-PerCP/Cy5.5 (BioLegend; [0.5 μg/ml]), α-Ly6G-APC (BioLegend; [1 μg/ml]), α-CD3e-APC (eBioscience; [1 μg/ml]), α-CD19-APC (eBioscience; [1 μg/ml]), α-CD49b-APC (eBioscience; [1 μg/ml]), and DAPI (Sigma; [0.1ug/ml]). Samples were stained concurrently with fluorescence minus one (FMO) antibody panels. Following a series of washes, the cells were resuspended in MACS buffer, and sorted on the BD Influx Cell Sorter (Becton, Dickinson and Company, Franklin Lakes, NJ). FlowJo software (Tree Star Inc., v.9.2, Ashland, OR) was used for data analysis.

On study day 85, all mice were anesthetized with 0.5 mg/kg Medetomidine, 5.0 mg/kg Midazolam i.p. and 0.05 mg/kg Fentanyl i.p., injection volume 0.1 ml/10 g of body weight, and blood was taken via cardiac puncture and anticoagulated with dipotassium ethylenediaminetetraacetic acid (K2EDTA). Immediately after, animals were euthanized via cervical dislocation. To calculate the N/L ratio whole blood was stained with respective antibodies for 15 min and after 15 min fixation with 1% formaldehyde, red blood cells were lysed with BD FACS Lysing Solution (BD Biosciences, San Jose, CA, USA). Afterward, the cells were centrifuged for 5 min at 500 × g, and the supernatant was discarded. The pellet was re-suspended in 1% formaldehyde, acquired by flow cytometry (Attune^® NxT Acoustic Focusing Cytometer, Thermo Fisher Scientific, Austria, Vienna) and analyzed by Attune™ NxT Software (Thermo Fisher Scientific, Austria, Vienna). The following anti-mouse antibodies were used: α-CD3e-AF488, α-CD11b-PerCP, α-B220-BV421, α-Ly6G-APC, α-CD45-BV650, (all Biolegend, San Diego, CA, USA). Neutrophils were defined as CD45⁺, CD11b⁺ Ly6G⁺ cells, lymphocytes were identified by CD45⁺, CD11b^– and verified by positivity for either B220 or CD3e.