Cells were stained with fluorochrome-labeled mAbs and analyzed with an LSRII flow cytometer (BD). Flow cytometric analysis was performed using FlowJo software. The following fluorochrome-labeled mAbs were purchased from Biolegend, Inc. and used according to the manufacturers’ protocols: anti-CD11b-FITC (101205, RRID: AB_312788), anti-CD206-APC (141707, RRID: AB_10896057), anti-Ly6C-APC-Cy7 (128026, RRID: AB_10640120), anti-Ly6G-PE (127608, RRID: AB_1186099), anti-F4/80-Brilliant Violet 421 (123132, RRID: AB_11203717), anti-CD64-PE (139303, RRID: AB_10613467), and anti-Siglec-F-APC (155507, RRID: AB_2750236).
Anti cd64 pe
Manufactured by BioLegend
Sourced in Germany, United States
Anti-CD64-PE is a monoclonal antibody that recognizes the CD64 antigen and is conjugated to the fluorescent dye R-Phycoerythrin (PE). CD64 is a high-affinity Fc gamma receptor expressed on the surface of monocytes, macrophages, and neutrophils. This antibody can be used to identify and quantify these cell types in flow cytometry applications.
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Lab products found in correlation
Anti cd206 apc, by BioLegend (2 mentions)
Anti cd45 pacific blue, by BioLegend (2 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (2 mentions)
Anti nk1.1 bv650, by BioLegend (2 mentions)
Anti b220 bv650, by BioLegend (2 mentions)
Anti mhc 2 af700, by BioLegend (2 mentions)
Zombi nir live dead stain, by BioLegend (2 mentions)
Fcr blocking antibody, by Miltenyi Biotec (2 mentions)
Anti cd11b bv605, by BioLegend (2 mentions)
Anti cd11c pe cy7, by BioLegend (2 mentions)
Anti ly6c percp cy5, by BioLegend (2 mentions)
Anti cd3 bv650, by BioLegend (2 mentions)
Lsrii cytometer, by BD (2 mentions)
Anti ly6c apc cy7, by BioLegend (1 mentions)
Anti f4 80 brilliant violet 421, by BioLegend (1 mentions)
Anti siglecf apc, by BioLegend (1 mentions)
Anti cd11b fitc, by BioLegend (1 mentions)
Anti ly6g pe, by BioLegend (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Anti f4 80 fitc, by BioLegend (1 mentions)
5 protocols using anti cd64 pe
1
Multiparametric Flow Cytometry Analysis
2
Multiparameter Profiling of Immune Cells
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THP-1 macrophages and bone marrow-derived macrophages were detached using ice-cold PBS, 0.1% EDTA. Lamina propria cells, peritoneal immune cells and lung immune cells were isolated as described previously.13 (link),44 (link),45 The cells were then incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 min and stained with anti-CD45-Pacific Blue, anti-CD3-BV650, anti-NK1.1-BV650, anti-B220-BV650, anti-CD11b-BV605, anti-CD11c-PECy7, anti-Ly6C-PerCPCy5.5, anti-F4/80-Fitc, anti-CD64-PE, anti-MHC-II-AF700, anti-CD206-PE-TexasRed, anti-pSTAT6-APC (all from BioLegend, mouse cells) anti-CD124-PE, anti-CD206-APC, (all from Biolegend, human cells) for 15–30 min. ZOMBI-NIR live dead stain (BioLegend, San Diego, CA) was used for discrimination between live and dead cells in all experiments. For pSTAT6 staining, cells were permeabilized using the FoxP3 staining kit from eBioscience. Samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc. Ashland, OR). For RNA expression analysis of lung immune cell susbsets, lung homogenates were sorted on a MoFlo Asterios EQ cell sorter (Beckman Coulter Life Sciences, Krefeld, Germany).
3
Isolation and Phenotyping of Human Neutrophils
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Peripheral blood mononuclear cells (PBMCs) were isolated via centrifugation on a Ficoll–Paque PLUS gradient (GE Healthcare, Uppsala, Sweden). Neutrophils were further isolated after dextran sedimentation and hypotonic lysis as previously described (20 (link)).
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.
Fresh EDTA anti-coagulated peripheral blood (100 μl) was incubated with antibodies for 30 min in the dark. The following antibodies were used: Anti-CD66b-PE-cy7, anti-TLR2-FITC, anti-TLR4-APC, anti-CXCR1-APC, anti-CXCR2-FITC, anti-C5aR-APC, anti-CD64-PE, anti-CD177-APC, anti-CD62L-FITC, anti-CD11b-PE, anti-CD49d- FITC, anti-C5L2-PE, anti-CD3-BV421 and anti-PD-1-PE were obtained from Biolegend (San Diego, California, USA); anti-CD4-BV421, anti-CD8-APC-cy7, anti-CD38-FITC, and anti-HLA-DR-PerCP were purchased from BD Biosciences (Franklin Lakes, New Jersey, USA); anti-PD-L1-PE were bought from eBioscience (San Diego, California, USA). Isotype-matching antibodies were used as negative controls. After lysing the red blood cells, the remaining cells were washed and fixed for flow cytometry analysis.
4
Comprehensive Flow Cytometry Analysis of Immune Cells
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For flow cytometry of immune cells, lamina propria immune cells were isolated as described.83 (link) For analysis of myeloid immune cells, the cells were washed in PBS, incubated with FcR blocking antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes, and stained with anti-CD45–Pacific Blue, anti-CD3–BV650, anti-NK1.1–BV650, anti-B220–BV650, anti-CD11b–BV605, anti-CD11c–PECy7, anti-Ly6C–PerCPCy5.5, anti-F4/80–APC, anti-CD64–PE, and anti–MHC-II–AF700 (all from BioLegend, San Diego, CA) for 15–30 minutes. ZOMBI-NIR live dead stain (BioLegend) was used for discrimination between live and dead cells. For cytokine staining, the cells were incubated with ionomycin and PMA in the presence of Brefeldin A for 3.5 hours before surface staining with anti-CD25–AlexaFluor700, anti-CD3–PerCPCy5.5, anti-CD4–BV510, and anti-CD8–BV570 for 15 minutes. Cells then were fixed with the FoxP3 staining kit (eBioscience) according to the manufacturer’s instructions, stained with anti-FoxP3–Pacific Blue, anti–IFN-γ–PECy7, anti-IL17–APC, anti-TNFα–BV650, and anti-IL22–PE for 30 minutes, washed in PermWash buffer (eBioscience), samples were acquired on an LSRII cytometer (BD, Franklin Lakes, NJ), and analyzed using FlowJo (Tree Star, Inc, Ashland, OR). Gating strategy for T and myeloid cells was performed as described previously.32
5
Surface Staining for Flow Cytometry
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For surface staining, cells were incubated with 50 µl purified anti-mouse CD16/32/FcBlock for 10 min at 4 °C, as described previously45 (link). Additional antibodies were then added in FACS buffer to a final volume of 100 µl at 4 °C for 20 min. After staining, cells were washed twice and resuspended in FACS buffer for analysis. Dead cells were excluded from analysis by fixable viability dye eFluor™ 780 (Thermo Fisher Scientific, MA). Flow cytometry was performed on an LSR Fortessa (BD Biosciences, NJ) with subsequent data analysis using FlowJo software (Tree Star). Cells were quantified by using CountBright Absolute Counting Beads (Thermo Fisher Scientific, MA). The following antibodies were purchased from Biolegend: anti-CD45.2-R-phycoerythrin-cyanine 7 (PECy7) (clone: 104), anti-MHCII I-A/I-E-AF700 (clone: M5/114.15.2), anti-CD11c-BV786 (clone: N418), anti–CD3e-PECy5 (clone: 145-2C11), anti-CD19-BV650 (clone: 6D5), anti-Ly6G-Peridinin-chlorophyll (PerCP)-Cy5.5 (clone: 1A8), anti-F4/80-AF647 (clone: BM8), anti-CD24-BUV395 (clone: M1/69), anti-CD64-PE (clone: X54-5/7.1), anti-Ly6C-BV605 (clone: HK1.5). The following antibodies were purchased from BD Biosciences: anti-CD11b-Brilliant UltraViolet (BUV) 737 (clone: M1/70).
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