Formalin-fixed and paraffin-embedded tissue sections were stained with anti-CD66b (305102, Biolegend), anti-cleaved caspase-3 (559565, BD Pharmingen, Franklin Lanes, NJ, USA), M30 (12140322001, Roche, Merck, Darmstadt, Germany), anti-IL-8 (MA5-23697, Thermo Fisher Scientific), anti-CD3 (MA5-14524, Thermo Fisher Scientific), anti-CD68 (M087629-2, Dako, Agilent Technologies, Carpinteria, CA, USA) and anti-CD206 (ab64693, Abcam, Cambridge, UK) antibodies for 1 h, followed by nuclear counterstaining with Hematoxylin Gill III (1.05174.0500, Merck). See supplementary material for extended protocol.
Anti cd66b
Manufactured by BioLegend
Sourced in United States
Anti-CD66b is a monoclonal antibody that binds to the CD66b antigen, which is expressed on the surface of granulocytes, including neutrophils, eosinophils, and basophils. It is a useful tool for the identification and enumeration of these cell types in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
M087629 2, by Agilent Technologies (2 mentions)
Anti cd11b, by BioLegend (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Anti cd45, by Thermo Fisher Scientific (2 mentions)
Ab64693, by Abcam (1 mentions)
Anti cd68, by Agilent Technologies (1 mentions)
Hematoxylin gill 3, by Merck Group (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Anti il 8, by Thermo Fisher Scientific (1 mentions)
Ma5 14524, by Thermo Fisher Scientific (1 mentions)
Anti cleaved caspase 3, by BD (1 mentions)
Easysep direct human neutrophil isolation kit, by STEMCELL (1 mentions)
1300exi cytometer, by Stratedigm (1 mentions)
Perm wash, by BD (1 mentions)
Anti epcam, by Abcam (1 mentions)
Trop2, by BioLegend (1 mentions)
Anti cd34, by BioLegend (1 mentions)
Anti pan cytokeratin, by BioLegend (1 mentions)
Anti androgen receptor, by Cell Signaling Technology (1 mentions)
Anti trop2, by BD (1 mentions)
11 protocols using anti cd66b
1
Immunohistochemical Profiling of Tissue
2
Neutrophil Phagocytosis Assay for SHIV Antigens
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The ADNP assay was adapted from Karsten et al. (40 (link)); gp140 SHIVSF162p3 and the recombinant Mymetics rgp41 antigens were coupled to beads and immune complexes were formed as described for ADCP. Neutrophils were isolated from fresh whole ACD-anticoagulated blood using EasySep Direct Human Neutrophil Isolation kit (Stem Cell, 19666), resuspended in R10 medium, and added to plates at a concentration of 5x104 cells/well. The plates were incubated for 30 min at 37°C. The neutrophil marker CD66b (Pacific Blue conjugated anti-CD66b; BioLegend, 305112) was used to stain cells. Cells were fixed for 10 min in 4% PFA. Fluorescence was acquired with a Stratedigm 1300EXi cytometer and phagocytic score was calculated as described for ADCP.
3
Identification and Characterization of Circulating Tumor Cells
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CTCs were assessed by immunofluorescence staining. The following antibodies were used to identify CTCs and assess EPCAM and TROP-2 staining as indicated in the figure legends: Anti-CD45 (BioLegend Cat# 304018, RRID:AB_389336), Anti-CD34 (BioLegend Cat# 343508, RRID:AB_1877133), Anti-CD11B (BioLegend Cat# 101218, RRID:AB_389327), Anti-CD66B (BioLegend Cat# 305109, RRID:AB_2563170), Anti-Pan cytokeratin (BioLegend Cat# 628602, RRID:AB_439775 or Abcam Cat# ab49779, RRID:AB_869395), Anti-Androgen Receptor (Cell Signaling Technology Cat# 5153S, RRID:AB_10692774), Anti-EPCAM (Abcam Cat# ab112068, RRID:AB_10861805) or Anti-TROP-2 (BD Biosciences Cat# 940370, RRID:AB_2876239) and Hoechst 33342 (Thermo Fisher Scientific). Extracellular antibodies were stained at 4°C for 30 minutes. For intracellular and nuclear staining of cells (Fig. 2 ), cells were stained as described by Sperger and colleagues (30 (link)). For intracellular staining (Fig. 4 ), cells were permeabilized, stained, and washed with BD Perm/Wash. Images were taken with a 10x objective using Nikon Eclipse Ti-E with an ORCA-Flash 4.0 V2 Digital CMOS camera (Hamamatsu) and NIS-Elements AR Microscope Imaging Software (RRID:SCR_014329, Nikon Instruments). Images were background subtracted, and CTCs were determined by Hoechst-positive staining, cytokeratin+ and CD45−/CD34−/CD66b−.
4
Measuring Immune-Complex Uptake in Granulocytes
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The ADNP assay was performed as previously described GCLP qualified assay that quantifies immune-complex uptake into cells (46 (link)) that was previously associated with protection from SIV infection in non-human primates (38 (link)). Briefly, whole blood was collected in ACD tubes. Granulocytes were isolated by lysing erythrocytes with ACK lysis buffer (Thermo Fisher Scientific, MA, USA) for 5 min before precipitation by centrifugation. Granulocytes were washed twice with PBS and were resuspended at 2.5 × 105 cells/ml in R10 and 50,000 cells per well were incubated with immune complexes for 1 h at 37°C, 5% CO2. Neutrophils were stained with anti-CD66b (Biolegend, San Diego, CA), and cells were fixed with 4% paraformaldehyde prior to flow cytometry. Phagocytosis scores were calculated as above in the ADCP assay.
5
Immunohistochemical analysis of SARS-CoV-2 infection
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Formalin-fixed paraffin-embedded lung tissue was cut into 4 μm sections and mounted on frosted glass slides. After deparaffinization and rehydration, slides were submerged into pH 8.0 EDTA buffer and boiled for 2 min with high pressure for antigenic retrieval. Slides were washed in PBS, incubated in 3% hydrogen peroxide for 10 min and then blocked with 1% bovine serum albumin (BSA) for 1 h. The following primary antibodies were used: anti-p-hRIPK1 (Cell Signaling Technology, 44590), anti-p-mRIPK1 (BioLynx), anti-RIPK1 (BioLynx, Clone 59), anti-p-MLKL (Abcam, ab187091), anti-p-RIPK3 (Cell Signaling Technology, 93654), anti-CD68 (Zymed, San Francisco, CA) and anti-SARS-CoV/SARS-CoV-2 nucleoprotein (Sino Biological, 40143-T62 and MM05), anti-CD66B (Biolegend, 305102), anti-cleaved Caspase-3 (Cell Signaling Technology, 9661). Slides were then washed in PBST (PBS plus 0.1% Tween 20), incubated with the biotinylated secondary antibody (Zymed, San Francisco, CA) and visualized with 3,3′-diaminobenzidine (DAB) under the microscope.
6
Multicolor Immunofluorescence Tissue Staining
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Formalin-fixed and paraffin-embedded tissue sections were incubated with anti-CD66b (305102, Biolegend), M30 (12140322001, Roche) and anti-CD68 (M087629-2, Dako) primary antibodies, followed by secondary antibody staining using donkey anti-mouse IgG-AF555 (A31570, Thermo Fisher Scientific) and goat anti-mouse IgM-DyLight650 (SA5-10153, Thermo Fisher Scientific). Nuclei were stained with DAPI (33342, Invitrogen) and sections were visualised using a LSM700 fluorescence microscope (Zeiss, Vienna, Austria). For extended protocol, see supplementary material.
7
Enriching Annexin A1+ Neutrophils for NET Imaging
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Due to the low proportion of Annexin A1+ neutrophils in the pan neutrophils, Annexin A1+ neutrophils were first enriched using FACS. After enrichment, neutrophils were stained with anti-CD66b (Biolegend) and anti-Annexin A1-APC (AssayLite) as described above in “Immunofluorescence staining for detection of NETs in neutrophils”. Images were obtained with a confocal microscope (Nikon).
8
Isolation of Macrophage Subsets from Colorectal Liver Metastases
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Macrophages were FACS sorted from surgically resected CLM tissues from five patients. Single-cell suspensions were obtained by manually mincing tissue into small fragments and incubating the fragmented tissues for 1 h at 37°C in HBSS (Euroclone) containing 1 mg/ml Type IV Collagenase (Sigma-Aldrich), 2% FBS (Sigma-Aldrich), 50 μg/ml DNase I (Sigma-Aldrich), and 10 mM Hepes (Lonza). The resulting cell suspension was filtered through a 70-μm cell strainer and erythrocytes were lysed with ACK Lysing Buffer (Lonza). Cells were then incubated with a blocking solution containing 1% human serum in saline solution and stained with the following fluorophore-conjugated primary antibodies: anti-CD45 (BD Biosciences; clone HI30), anti-CD11b (BD Biosciences; clone ICRF44), anti-CD16 (BioLegend; clone 3G8), anti-CD14 (BD Biosciences; clone M5E2), anti-CD66b (BioLegend; clone G10F5), and anti-CD163 (BD Biosciences; clone GHI/61). Fixable Viability Stain 700 fluorescent dye (BD Biosciences) was used to perform dead cell exclusion. SYTO 16 Green Fluorescent Nucleic Acid Stain (ThermoFisher) was used to identify nucleated cells. Large and small macrophages were FACS sorted on a FACSAria III (BD Biosciences) following the gating strategy shown in Fig. S2 .
9
Neutrophil Identification by Flow Cytometry
10
Multiparametric Analysis of Tumor Immune Microenvironment
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Analysis of tumour cell death was performed following incubation of cells with Zombie Yellow dye (423103, Biolegend) and intracellular active caspase-3 staining (559565, BD Pharmingen). For immune cell phenotyping, cells were stained using the following antibodies: anti-CD62L (17-0626, eBioscience, Thermo Fisher Scientific, San Diego, CA, USA), anti-CD66b (305104, Biolegend), anti-CD11b (301310, Biolegend), anti-CD16 (MHCD1617, eBioscience), anti-CD206 (321138, Biolegend), anti-CD86 (305406, Biolegend), anti-CD14 (301814, Biolegend), anti-HLA-DR (307618, Biolegend), anti-CD163 (17-1639-42, eBioscience), anti-CD45 (48-0459-41, eBioscience), anti-CD3 (17-0037-42, eBioscience). To assess neutrophil apoptosis, cells were stained with anti-CD15 (12-0159, eBioscience), annexin V and 1 µg/ml propidium iodide (V13242, FITC Annexin V/Dead Cell Apoptosis Kit, Invitrogen,). Sample acquisition was performed on a Gallios Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) and data was analysed using the Kaluza 2.1 software (Beckman Coulter). See supplementary material for extended protocol.
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