Anti cd115 pe

Peripheral blood samples were used for the detection of monocyte subsets upon clodronate treatment. For the analysis of TSLP expression in the skin, excised tissue samples were placed into a cocktail of collagenase I, collagenase XI, DNase I and hyaluronidase (Sigma‐Aldrich); and shaken at 37°C for 1 hour. Cells were then triturated and centrifuged (15 minutes, 500 g, 4°C).
Cells were labelled with anti – CD11b – FITC or eFluor 450 (eBioscience), anti–Gr1‐PerCP‐Cy5.5 (eBioscience), anti‐CD115‐PE (eBioscience), anti–CD11c‐PE‐Cy7 (eBioscience) or anti–CD45‐FITC (eBioscience) and then analysed by flow cytometry on a Fortessa cytometer (Becton Dickinson). For intracellular cytokine staining, surface staining was performed before permeabilization using an intracellular staining kit (eBioscience). TSLP was labelled with anti‐TSLP‐Alexa Fluor 488 (Bioss Antibodies).

We collected 100-µl aliquots of tail blood into EDTA-containing tubes. Cells in some tubes were Fc-blocked with 1 µg of mouse IgG (105 cells) for 15 min at room temperature without excess washing. After blocking, cell suspensions were then labeled with several monocyte-surface markers (anti-CD115–PE, eBioscience, catalog #17-1152; and anti-Ly-6C–APC, eBioscience, catalog #12-5932) and anti-CCR2–FITC (B&D, catalog: FAB5538F) or anti-CX3CR1-FITC (catalog #FAB5825G, R&D Systems, Minneapolis, MN, USA) at 4°C for 30 min. After cells were lysed by the addition of 4 ml Whole Blood Lysing Reagent (catalog #FC002, R&D Systems), the cells were washed twice with ice-cold PBS followed by centrifugation at 300× g for 5 min. The pellets were resuspended by adding 150 µl PBS. Controls included cells incubated with fluorochrome-conjugated unspecific isotype Ab as needed. Cell-surface molecule expression measurement and cell sorting were performed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Acquired fluorescence-activated cell sorting (FACS) data files were analyzed using the flow cytometry CellQuest software (BD Biosciences). Blood CD115+/Ly-6high cells defined as monocytes were further analyzed for CX3CR1 and CCR2 expression.