Podoplanin pe

For FACS analysis in vitro cultured cells and tissues were prepared as previously described (12 (link)). In the case of the lung, tissue was digested in collagenase IV-containing Grey's Balanced Salt Solution (GBSS, Sigma-Aldrich). Cell suspensions were stained with anti-mouse CD31-APC, anti-mouse CD45-PerCP and podoplanin-PE (all from Biolegend) to identify LECs (CD45⁻CD31⁺podo⁺) and BECs (CD45⁻CD31⁺podo⁻), respectively. ALCAM expression was detected by staining with goat anti-mouse ALCAM antibody (R&D Systems) and corresponding AlexaFluor488- or PE-labeled secondary antibodies (Invitrogen). Some FACS experiments were performed using I/F8-Fc (2 μg/ml) or KSF-Fc (2 μg/ml) followed by incubation with anti-mouseAlexaFluor488 or -PE-labeled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Data were acquired on a BD FACSCanto (BD Bioscience, Franklin Lake, NJ, USA) using FACSDiva software (BD Bioscience) and analyzed with FlowJo software 8.7.1. (Treestar, Ashland, TN, USA).

Single cell suspensions from mouse experiments were stained with Live/dead Aqua (Molecular Probes, Inc.), CD4-PerCP, CD8-Pacific Blue, CD69-BV605, CD62E-PE, CD62E-BV421, CD62P-Alexa647, CD162-AlexaFlour647, HECA454-PE (BD Bioscience), CD25-APC, CD31-BV605, CD105-Pacblue, I/A-I/E-BV421, CD45-APCCy7, EpCAM-APC-Cy7, Podoplanin-PE, CD31-PECy7, EpCAM-FITC, CD64-BV711 (Biolegend), CD11c-PECy5.5 (Invitrogen) CXCR3-PECy7, CD3-Alexa700 and Ly6C-efluor450 (eBioscience). For subsequent detection of CXCL10 mRNA Primeflow^® RNA Assay (eBioscience) was used accordingly to manufacture protocol. Samples were acquired on an LSR-II flow cytometer (BD Biosciences) and analysed using FlowJo software (Tree Star Inc.).
Single cell suspension isolated from human tumors and unaffected colon tissue were stained with Live/dead Aqua (Molecular Probes, Inc.), CD31-Alexa700, (Biolegend), CD4-PerCP, CD8-BV711, CD105-APC, CD14-Alexa700, CD19-APCH7 and CD19-PE-CF594 (BD bioscience) followed by permeabilization with Fix & Perm kit (ADG Bio research GMBH) and staining with CXCL9-FITC (R&D) and CXCL10-PE (Biolegend), flow cytometry analyses were performed as above.