Single cell suspensions from surgical excess colon tissue or HIV-uninfected control PBMCs were stained for viability with a LIVE/DEAD fixable violet dead cell stain kit (Life Technologies). For phenotypic characterization of epithelial and tissue cells, cell surface antigens were stained as described above with the following mouse anti-human monoclonal antibodies: APC anti-CD326 (clone EBA-1; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience). For phenotypic characterization of monocytes and DCs from PBMCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD66b (clone G10F5; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). Stained cells were resuspended in PBS with 5 mM EDTA and sorted using a FACS Aria II (BD Biosciences).
Cd66b clone g10f5
Manufactured by BD
Sourced in United States
CD66b (clone G10F5) is a cell surface antigen expressed on human granulocytes, including neutrophils, eosinophils, and basophils. It is a member of the CD66 family of cell adhesion molecules and plays a role in cell-cell and cell-matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd11c apc clone b ly6, by BD (2 mentions)
Anti cd3 fitc clone ucht1, by BioLegend (2 mentions)
V500 anti cd45 clone hi30, by BD (2 mentions)
Apc h7 anti cd16 clone 3g8, by BD (2 mentions)
Af700 anti hla dr clone l243, by BD (2 mentions)
Pe cy7 anti cd123 clone 7g3, by BD (2 mentions)
Cd19 clone hib19, by BioLegend (2 mentions)
Facsaria 2, by BD (1 mentions)
Live dead fixable violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Percp cy5.5 anti cd14, by BioLegend (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Cd56 clone hcd56, by BioLegend (1 mentions)
Cd8 clone c8 144b, by Agilent Technologies (1 mentions)
Cd63 clone h5c6, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Apc cd33 clone wm53, by BD (1 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Ba310, by Motic (1 mentions)
Facscanto flow cytometer, by BD (1 mentions)
7 protocols using cd66b clone g10f5
1
Phenotypic Characterization of Colonic and Immune Cells
2
Phenotypic Profiling of Cryopreserved PBMCs
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Cryopreserved PBMCs from acute (n = 8) and HIV-uninfected control subjects (n = 9) were thawed and 0.5x106 cells were stained for viability with a LIVE/DEAD fixable blue or green dead cell stain kit (Life Technologies). For phenotypic characterization of monocytes and DCs, cell surface antigens were stained for 15 min at room temperature with the following mouse anti-human monoclonal antibodies: FITC anti-CD3 (clone UCHT1; Biolegend), CD19 (clone HIB19; Biolegend), CD56 (clone HCD56; Biolegend), CD66b (clone G10F5; BD Bioscience), BV605 anti-CD4 (clone SK3; BD Bioscience), v500 anti-CD45 (clone HI30; BD Bioscience), APC-H7 anti-CD16 (clone 3G8; BD Bioscience), PE-Cy5 anti-CXCR4 (clone 12G5; BD Bioscience), AF700 anti-HLA-DR (clone L243; BD Bioscience), APC anti-CD11c (clone B-ly6; BD Bioscience), PE-Cy7 anti-CD123 (clone 7G3; BD Bioscience), BV421 anti-CCR5 (clone 2D7; BD Bioscience), PerCP-Cy5.5 anti-CD14 (clone M5E2; Biolegend). The cells were fixed with 2% paraformaldehyde before running on a LSR Fortessa flow cytometer (BD Biosciences) within 4 h. Flow data were analyzed with FlowJo (TreeStar).
3
Prostate Tissue Microarray for Immune Cell Analysis
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We retrieved separate 51 RP specimens obtained at Yokohama City University Hospital (Yokohama, Japan). Appropriate approval from the institutional review board at our institution was obtained before the construction and use of the TMA. This was constructed from each representative lesion (benign and carcinoma). The clinicopathologic characteristics of these 51 patients are summarized in Table 2 .
IHC was performed on the sections (5 µm thick) from the prostate TMA, as described previously [7 (link)], using a primary antibody to CD66b (clone G10F5, diluted at 1 : 200, BD Biosciences, San Jose, CA, USA) or CD8 (clone C8/144B, diluted at 1 : 100, DAKO Corporation, Carpinteria, CA, USA). The slides were examined by a single pathologist (Hiroshi Miyamoto) blinded to the sample identity. The total numbers of CD66b-positive and CD8-positive cells were counted in each TMA core.
IHC was performed on the sections (5 µm thick) from the prostate TMA, as described previously [7 (link)], using a primary antibody to CD66b (clone G10F5, diluted at 1 : 200, BD Biosciences, San Jose, CA, USA) or CD8 (clone C8/144B, diluted at 1 : 100, DAKO Corporation, Carpinteria, CA, USA). The slides were examined by a single pathologist (Hiroshi Miyamoto) blinded to the sample identity. The total numbers of CD66b-positive and CD8-positive cells were counted in each TMA core.
4
Neutrophil Phenotype and Activation
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For analysis of phenotype, freshly isolated cells were incubated with the CD15 (clone HI98), CD16 (clone NKP15), and CD 62L (clone DREG-56) to find out surface expression of those markers. In addition, to find degranulation and activation status of neutrophils, the expression of various granule markers such as CD 35 (clone E11), CD63 (clone H5C6), and CD 66b (clone G10F5) antibodies (BD Biosciences, San Jose, CA, USA) was evaluated at 4°C for 30 min in the dark. Cells were then washed with PBS twice and resuspended in 0.5 mL of 0.5% paraformaldehyde (Sigma Chemical Co, Poole, UK). Flow cytometry was performed using a FACScalibur (BD Biosciences, San Jose, CA, USA) based on CD15-positive neutrophils. For all cases, at least 15,000 cells were acquired for each case and analyzed by gating on neutrophils.
5
Characterization of Neutrophil Functionality
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At the end of granulopoietic differentiation, cells were cytospun onto a Superfrost Plus Microscope slide (Fisherbrand, ThermoFisher Scientific, Waltham, MA). The slides were Wright–Giemsa stained and scored for myeloid cell types (promyelocytes, myelocytes, metamyelocytes, bands, neutrophils, and monocytes) using an upright microscope (Motic BA310). For the immunophenotypic characterization of neutrophils, cells were stained for CD45‐Pacific Blue (clone HI30, catalog #560367), CD34‐PECy7 (clone 581, catalog #561440), CD33‐APC (clone WM53, catalog #551378, CD11b‐5APCCy7 (clone ICRF44, catalog #557754, clone ICRF44), and CD16‐PE (clone W6D3, catalog #562370), and CD66b (clone G10F5, catalog #561927) from BD Biosciences. The neutrophil population (defined as CD45+/CD34−/CD14−/CD11b+/CD16+) was gated and used for analysis of ROS generation, bacterial phagocytosis and phospho‐FACS in a FACS‐Canto flow cytometer (BD Biosciences).
6
Multimodal Immunohistochemistry Analysis
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CRC tissues were fixed using neutral-buffered formalin and embedded in paraffin. The sections were stained with Perls’ reagent and developed using DAB, as previously described (42 (link)). After iron staining, the slides were subsequently incubated with primary antibodies against CD8 (clone SP16, ab9829; Abcam), CD66b (clone G10F5, 555723; BD Pharmingen), cytokeratin 20 (clone Ks20.8, 413491; Nichirei), and IBA1 (polyclonal, 019-19741; Wako) overnight at 4°C. The sections were visualized using HistoGreen (E109; Cosmo Bio) and counterstained with Mayer hematoxylin. CCL8 (clone 1.1_2D4-1A3, LS-B8198; LSBio) staining was conducted using DAB, followed by costaining with IBA1 using HistoGreen. Images were obtained with a KEYENCE BZ-X800 all-in-one microscope (KEYENCE). Quantification was performed using the KEYENCE BZ analyzer.
7
Neutrophil Subset Analysis in TEVAR
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Approximately 5 mL whole blood samples were taken at admission before TEVAR surgery. Peripheral leukocytes, whole neutrophils, monocytes and lymphocytes were evaluated by Reflotron Plus Hematology Analyzer (Roche, Swiss). Ethylenediaminetetraacetic acid (EDTA)-anticoagulated blood samples from enrollments were processed for flow cytometry to determine the number of neutrophil subsets as previously described. After incubated with Fc-block (BioLegend, USA), cells were stained with CD66b (clone G10F5, BD Bioscience, USA), CD11b (clone ICRF44, BD Bioscience, USA), CD14 (clone M5E2, BD Bioscience, USA), CD101 (clone BB27, BD Bioscience, USA) and CD10 (clone HI10a, BD Bioscience, USA) for 15 minutes at room temperature. FSC/SSC gate was positioned to exclude cell debris, then total neutrophils were identified by CD11b, CD66b and CD14. Human circulating neutrophils could be categorized into three subpopulations: a numerically dominant CD10+CD101+ mature neutrophils, CD10−CD101+ immature neutrophils and CD101− pre-neutrophils. FlowJo software (Version 10, USA) was conducted to analyze the data generated by flow cytometry.
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