Anti cd14 ecd

Colorectal tissue was digested with an enzyme cocktail including dispase I and DNase I, as previously described [23 (link)]. The resulting cellular suspension was washed in PBS, counted and stained for flow cytometry analysis at a concentration of 10⁶ cells/mL. Mononuclear cells (MNCs) were fixed in a PBS solution containing 2% paraformaldehyde, 60 mM sucrose at pH 7.4, for 15 min at room temperature. Fixation is necessary for MAb 45523 to bind to cell surface CCR5 on primary CD4⁺ T-cells. The fixed cells were washed twice with PBS containing 20 mM glycine (buffer A) and incubated for 15 min at room temperature in buffer A supplemented with 1% BSA and 0.05% NaN₃ (buffer B). Cells were then stained with mixtures of MAbs in buffer B, anti-CD4-APC-Cy7, anti-CD13-PE, anti-CCR5-2D7-FITC, anti-CXCR4-12G5-FITC, anti-HLA-DR-PE-Cy7 (BD Pharmingen, San Diego, CA), anti-CCR5-45531-FITC, anti-CCR5-45523-FITC, anti-DC-SIGN-PE (R&D Systems), anti-CD14-ECD, anti-CD64-PE, or anti-89-PE (Beckman Coulter), for 1 h at room temperature. After three washes in buffer B, cells were resuspended for analysis using a BD LSR II flow cytometer (BD Biosciences; San Jose, CA, USA). The parameters used to select cell populations for analysis were forward and side-light scatter, with a total of 10,000 events being collected for analysis as described previously [24 (link)].

Fresh PBMCs were isolated from whole blood by density-gradient centrifugation using the BD Vacutainer CPT Mononuclear Cell Preparation Tubes with Sodium Heparin (BD Biosciences, Franklin Lakes, NJ, USA). The cells were first stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) and then with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD11c-Alexa700, anti-HLADR-V500, anti-CD19-APC-H7 (all from BD Biosciences), anti-CD14-ECD, anti-CD56-APC, (both from Beckman Coulter, Brea, CA, USA), anti-CD123-FITC, anti-CD3- PerCPCy5.5, anti-CD56-BV421 (all from BioLegend), and anti-CD19-PE (TONBO Biosciences, San Diego, CA, USA). pDCs were identified as CD3^-CD19^-CD14^-CD56^-HLADR⁺CD11c^-CD123⁺(Additional file 2: Figure S1). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentages of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).