Ly 6c apcefluor780

To obtain a single cell suspension, spleen was gently disaggregated with a pestle and then with a syringe in 1 ml PBS1x, 2 mM EDTA, after letting debris decant for few minutes, the superior phase was transferred in a new tube. The following procedure was applied to both spleen single cell suspensions and blood samples: after erythrocyte depletion with ACK lysis buffer, 10⁶ cells were suspended in 100 μl RPMI, 10 mM Hepes, 1 mM EDTA, 2% FBS and labeled for 30′ at RT with 1 μg of the following antibodies: CD4-APC (eBioscience, SanDiego, CA, USA), CD8-PeCy7 (eBioscience), CD11b-FITC (eBioscience), B220-PacificBlue (BioLegend, San Diego, CA, USA), Ly-6G-PE (BioLegend), Ly-6C-APCeFluor780 (eBioscience). After labeling cells were washed twice with PBS1x, suspended in 200 μl PBS1x 2 mM EDTA and analyzed with BD LSR II Flow Cytometer (BD Biosciences, San Jose, CA, USA).

The following anti-mouse mAbs were used for surface staining during flow cytometry: CD3-eFluor 450, CD8-PerCP-Cy5.5, CD11b-AlexaFluor 700, CD11b-eFluor 450, CD14-FITC, CD25-APC, CD25-eFluor 450, CD45.1-APC, Ly6C-APC-eFluor 780, I-A/E-FITC (eBioscience, San Diego, CA, USA), CD45.1-eFluor 450, CD45R-Horizont V500, CD3-Horizont V500, CD4-Horizont V500, CD4-PerCP, CD8-Horizont V500, Ly6G-AlexaFluor 700, Siglec F-PE (BD Biosciences, San Jose, California, USA) and TLR4-APC (BioLegend, San Diego, Ca, USA). For intracellular staining, the following anti-mouse mAbs were used: TNFα-PE, Foxp3- and IFNγ-PE (eBioscience; San Diego, California, USA). Fc-block (anti-CD16/CD32 mAbs; eBioscience, San Diego, California, USA) was used both in surface and intracellular staining. For ELISA, matched anti-mouse capture mAb and biotinylated detection mAb against TNF-α, IFN-γ, IL-1β, IL-12 and IL-6 were used (R&D System, Minneapolis, Minnesota, USA). Blocking anti-mouse CD25 (PC61.5), CD4 (GK1.5) and anti-IFN-γ (XMG1.2) mAbs, as well as anti-mIL-2 mAbs for preparing IL-2 complexes (S4B6, JES6-1A12), were obtained from BioXcell (Lebanon, New Hampshire, USA).