Anti gr1 percpcy5

On day 7, lungs were collected aseptically after mice were euthanized. Lungs were passed through 70 um cell strainer to obtain single-cell homogenates. Red blood cells were lysed and the remaining cells were washed with FACS buffer. After cells were resuspended in 200 ul of FACS buffer, Fc receptors were blocked with anti-CD16/CD32 (Fc Block, BD) and then stained using the following antibodies (all from eBiosciences): anti-CD11b-APC, anti-CD11c-PECy7, anti-CD45-AF700, anti-Gr1-PerCpCy5.5, viability-e520, anti-Ly6C-PE, anti-MHCII-e450, anti-SiglecF-PE-CF594.

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).