Small pieces of human spleen, liver or lung were embedded in Tissue Tek, frozen in liquid nitrogen vapor and stored at -80°C. Cryostat sections of 7 μm thickness were cut, fixed in acetone for 10 minutes and rehydrated in PBS. The sections were blocked with 5% human serum (Brocacef, Amsterdam, the Netherlands) in PBS for 15 minutes prior to 30 minutes staining with the following antibodies: anti-CD68 (clone Y1/82A, BD BioSciences), anti-VE-Cadherin (clone 55-7H1, BD BioSciences), anti-CD163 (clone MAC2-158, Trillium Diagnostics), anti-CD19 (clone HIB19, BioLegend), anti-CD64 (clone 10.1, BioLegend), anti-CD32a,b,c (clone AT10, Serotec), anti-CD32a (clone IV.3, SanBio), anti-CD32b,c (clone 2B6, a generous gift from MacroGenics, Rockville, MD) and anti-CD16 (clone 3G8, BioLegend). After washing with PBS, the sections were embedded in mowiol (Calbiochem). Isotype-matched antibodies were used as negative controls, i.e. the detection limit was increased until the level was reached at which the isotype control did not show any fluorescent signal anymore [18 (link)]. Stainings were analyzed using a DM6000 Leica immunofluorescence microscope or Leica SP8 confocal microscope.
Anti cd16 clone 3g8
Manufactured by BioLegend
Sourced in Moldova, Republic of, United States, United Kingdom
Anti-CD16 (clone 3G8) is a lab equipment product. It functions as an antibody that binds to the CD16 receptor, which is expressed on the surface of certain immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd19 clone hib19, by BioLegend (2 mentions)
Anti cd64 clone 10, by BioLegend (2 mentions)
Anti cd4 clone okt4, by BioLegend (2 mentions)
Anti cd14 clone m5e2, by BioLegend (2 mentions)
Lsr 2, by BD (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Anti cd68 clone y1 82a, by BD (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Cellsearch ctc kit, by Menarini Silicon Biosystems (1 mentions)
Cellsearch system, by Menarini Silicon Biosystems (1 mentions)
Human cd25 clone bc96, by BioLegend (1 mentions)
Human foxp3 clone 236a e7, by BioLegend (1 mentions)
Human pd 1 clone eh12.2h7, by BioLegend (1 mentions)
Anti cd14 clone hcd14, by BioLegend (1 mentions)
Anti cd56 clone hcd56, by BioLegend (1 mentions)
Anti cd3 clone hit3a, by BioLegend (1 mentions)
Fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Facsaria fusion, by BD (1 mentions)
Lab tek 2, by Thermo Fisher Scientific (1 mentions)
Rituximab, by GlaxoSmithKline (1 mentions)
12 protocols using anti cd16 clone 3g8
1
Immunofluorescence Staining of Frozen Tissue Sections
2
Detecting Circulating Tumor Cells using CellSearch
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Aliquots of 7.5 mL of blood were processed with the CellSearch® system (Menarini Silicon Biosystems, Castel Maggiore (BO), Italy) for CTC detection. CellSearch analysis was performed within 96 h after blood was drawn. Antibodies directed against the epithelial cell adhesion antigen (EpCAM) coupled to ferrofluids were used to enrich CTC from the background of leukocytes. The enriched cells were fluorescently labeled with the CellSearch CTC kit (Menarini Silicon Biosystems) using the nucleic acid dye 4′6-diaminodino-2-phenylindole (DAPI) for DNA staining, anti-cytokeratin monoclonal antibodies (mAbs) C11 and A53.B/A2 labelled with phycoerythrin (PE), and anti-CD45 mAb (clone HI30) labeled with allophycocyanin (APC) for recognizing leukocytes. Peridinin Chlorofyll A Protein (PerCP) labeled mAb anti-CD16 (clone 3G8, Biolegend, San Diego, CA, USA) directed against granulocytes, and Alexa-Fluor488 conjugated to the lectin wga (Thermo Fisher Scientific, Waltham, MA, USA) were added to the extra marker position in the CellSearch Epithelial Cell kit with an end concentration of 2 µg/mL and 3 µg/mL, respectively. This extra marker channel was used for the measurement of PerCP and DIOC on the CellTracks Analyzer II, which generates images of the complete cartridge for all channels.
3
Multiparameter Flow Cytometry Profiling of PBMCs
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Human PBMCs were stained with anti-CD14 (clone M5E2, Biolegend), anti-CD16 (clone 3G8, Biolegend), anti-CD4 (clone OKT4, Biolegend), human CD39 (clone 498403, R&D Systems), human CD73 (clone 606112, R&D Systems)44 (link),45 (link), human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236 A/E7, Biolegend), and human PD-1 (clone EH12.2H7, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR). Single stained samples were used to determine compensation values, and fluorescence minus one (FMO) controls were used to determine gating placement.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR). Single stained samples were used to determine compensation values, and fluorescence minus one (FMO) controls were used to determine gating placement.
4
Isolation and Sorting of cNK and NK Cells
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PBMCs were thawed, stained with LIVE/DEAD Green Viability Dye (Invitrogen) for 15 minutes at 4°C and subsequently preincubated for 10 min with of FcR blocking reagent (Miltenyi). Afterward, cells were incubated for 25 minutes at 4°C with anti-CD3 (clone HIT3a; BioLegend), anti-CD14 (clone HCD14; BioLegend), anti-CD19 (clone HIB19; BioLegend), anti-CD56 (clone HCD56; BioLegend) and anti-CD16 (clone 3G8; BioLegend) antibodies. Cells were then sorted for CD56dim CD16+ cNK cells and CD56bright CD16- NK cells on a BD FACSAria Fusion (BD Bioscience) at the Ragon Institute Imaging Core Facility.
5
Preparation of Eight-Chambered Glass Coverslips for NK Cell Binding Studies
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Preparation of eight-chambered glass coverslips was adapted from published protocols (Culley et al., 2009 (link)). Briefly, slides (1.5 Lab-Tek II; Nunc) were coated with 0.01% poly-L-lysine, dried, and coated with ligands for NK cells in PBS overnight at 4°C. 2.5 µg/ml MICA (Fc tagged; R&D Systems), 2.5 µg/ml MICA (His-tagged; Acro Biosystems), 10 µg/ml rituximab (GlaxoSmithKline), 2.5 µg/ml ULBP2 (Fc tagged; Abcam), 10 µg/ml anti-CD16 (clone 3G8; BioLegend), or 10 µg/ml anti-NKp30 mAb (clone P30-15; BioLegend), all with 2.5 µg/ml ICAM-1 (not tagged; R&D Systems) or 2.5 µg/ml Noggin (Fc tagged; R&D Systems), were used unless indicated otherwise. Where noted, 2.5 µg/ml ICAM-1 alone or 2.5 µg/ml of Noggin alone were also used as controls. In all experiments, recombinant human proteins MICA, ULBP2, and Noggin were used as Fc constructs and are referred throughout as MICA, ULBP2, and Noggin unless indicated otherwise. Slides were then washed with PBS and used for sample preparation.
6
Neutrophil Extracellular Trap Quantification
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Murine neutrophils were isolated following harvest of bone marrow from 8-week-old naïve B6 or PD-L1 KO mice. In brief, skin, muscle and fat were removed from femurs and tibias. Bone marrow was then flushed using a 27g needle and RPMI (Gibco). Following isolation of pure neutrophils, cells were then treated with 250-500 nM phorbol-12-myristate-13-acetale (PMA, Sigma-Aldrich) for 4 hours and then spun at 480xg for 5 minutes. The pellet was discarded and supernatant containing NET chromatin was spun at 18,000xg for 10 minutes. Quantification of NETs was analyzed using a Nano Drop (Thermo Fisher Scientific). In-vivo NETs quantification was analyzed using myeloperoxidase (MPO) (Roche) associated with DNA ELISA (Roche). For the human studies, fresh peripheral blood obtained from healthy donors or patients was stained with anti-CD15 (clone W6D3, BioLegend), anti-CD16 (clone 3G8, BioLegend), anti-citrullinated histone H3 (Abcam), anti-PD-L1 (clone 29E.2A3, Biolegend) and specific secondary anti-antibodies (Thermo Fisher Scientific).
7
Antibody Modulation of ZIKV Infection in PBMCs
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Freshly isolated PBMCs from healthy donors were first incubated with anti-CD16 (clone 3G8; Biolegend) or anti-CD32 (clone IV.3; GeneTex) or anti-CD64 (clone 10.1; GeneTex), respectively, at a range of antibody concentrations (1, 3, 10 μg/ml) or three antibodies combined (1, 3 or 10 μg/ml in total, equal amount of each antibody) for 1 h at 4°C. Mouse IgG isotype (GeneTex) was utilized as a negative control in the same range of concentrations (1, 3, 10 μg/ml). After incubation, cells were washed twice with RPMI-1640 medium followed by infection with ZIKV only (MOI 2) or ZIKV that had been pre-incubated with DENV immune serum complexes (MOI 2, D1-E01 serum sample at the dilution of 1/2,500) for 1.5 h at 37°C. Cells were subsequently washed once and cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin and 2 mM of L-glutamine at 37°C for 2 days. Following 2-day culture, 200 μl cell supernatants were harvested for further ZIKV amplification in Vero cells for an additional 2 days. Infected Vero cells were intracellularly stained with 4G2 antibody and the proportion of ZIKV infected cells was measured by flow cytometry.
8
Investigating Immune Checkpoint Proteins
9
Measuring Degranulation and Cytokine Production
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To study degranulation, PBMC were cocultured for 4 h with K562 cells. Anti-CD107a (Becton Dickinson (BD), FITC) was added to the PBMC at start of the coculture. Intracellular chemokine (CCL4, XCL1) and cytokine (IFN-γ) production was measured after stimulation with coated anti-CD16 for 4 h or K562 cells for overnight. For the CD16 stimulation, a flat-bottom plate was coated with 5 μg/ml goat anti-mouse antibody (BD) and subsequently with 1 μg/ml anti-CD16 (clone 3G8, Biolegend) in PBS. Mouse IgG1 isotype (1 μg/ml, Biolegend) served as negative control. Cells were cultured in AIM-V medium (Life Technologies, Bleiswijk, the Netherlands) supplemented with 10% FCS (Sigma–Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin (Sigma–Aldrich) at 37 °C. For each cell culture condition, after 1 h of culture Golgistop (BD) was added.
10
Monocyte Immunophenotyping by Flow Cytometry
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