Bb515 rat anti cd11b

Peripheral venous blood samples was subjected to density centrifugation using Ficoll-hypaque solution to isolate mononuclear cells and granulocytes. After lysing RBCs, cells were incubated with BB515 Rat anti-CD11b (BD Biosciences) and BV650 mouse anti-human CD16 (BD Biosciences) antibodies and sorted by flow cytometry using BD FACS AriaII. RNA sequencing (RNA-seq) was performed on sorted CD11b⁺CD16⁺myeloid cells in capecitabine-sensitive patients and CD11b⁺CD16⁻myeloid cells in capecitabine-resistant patients. Qubit 2.0 (Life Technologies, USA) and Bioanalyzer 2100 (Agilent, Germany) were used to analyze the RNA quality and integrity. A total of 3 μg RNA was used for the RNA sample preparations. Extracted RNA samples were processed using the NEBNext® UltraTM RNA Library Prep Kit (Lexogen) and sequenced on an Illumina Hiseq X-Ten with control of Hiseq Control Software (HCS). The sequencing library were qualified by Qubit 2.0 (Life technologies, USA) and Bioanalyzer 2100 (Agilent, Germany). Raw reads were processed through in-house perlscripts. Clean reads were obtained by removing reads containing adapter. Differentially expressed genes were defined by P < 0.05 and an absolute fold change > 2. Using Gene Set Enrichment Analysis, enrichment of a specific gene set was tested, and core enrichment genes were determined.

Peripheral venous blood samples were subjected to density centrifugation using Ficoll-hypaque solution to isolate mononuclear cells and granulocytes. Cell suspension was treated with BD Pharm Lyse lysing solution for red blood cell lysing. To determine the frequency of different subsets myeloid cell subsets, these cells (10^6 cells/tube) were stained by monoclonal antibodies BB515 Rat anti-CD11b (BD Biosciences, Clone # M1/70), PE Mouse anti-human CD66b (BD Biosciences, Clone # G10F5) or PE mouse IgM κ isotype control (BD Biosciences, Clone # G155–228), BV650 mouse anti-human CD16 (BD Biosciences, Clone # 3G8), PerCP-Cy5.5 mouse anti-human CD15 (BD Biosciences, Clone # HI98) or PerCP-Cy5.5 mouse IgM κ isotype control (BD Biosciences, Clone # G155–228), PE-Cy7 mouse anti-human CD14 (BD Biosciences, Clone # M5E2) and BV421 mouse anti-human CD33 (BD Biosciences, Clone # WM53) at 4 °C for 30 min respectively. Flow cytometry was performed on an LSRFortessa (BD Biosciences) and analyzed using FlowJo Version X (Tree Star, Inc).