Anti cd13 pe

Cells were stained with anti-CD31-PE, anti-CD33-FITC, anti-CD34-PE, anti-CD45-FTIC, anti-CD13-PE, anti-CD44-PE, anti-CD45-FITC, anti-CD71-PE, anti-CD90-PE, anti-CD95-APC, anti-HLA-ABC-FITC, anti-HLA-DR-FITC (all from BD Bioscience), anti-CD31-APC (eBioscience), anti-CD13-PE (Biolegend) and anti-CD105-FITC (R&D Systems). The stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson).

Cells at the third passage (P3) were digested with trypsin to form a single cell suspension solution, which was incubated with antibodies, including anti-CD34-PE, anti-CD31-APC, anti-CD45-PerCP-Cy5-5, anti-CD10-APC-Cy7 (BioLegend, USA), anti-CD13-PE, and anti-CD49d-PE-Cy7 (BD Biosciences, USA). All antibody incubations were performed at 37°C in the dark for 30 min. The cells were analyzed by flow cytometry (LSR II, BD Biosciences, USA) and Treestar FlowJo software.

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)].