All analytical flow cytometry was performed on a BD LSR II flow cytometer. Dead cells were identified by DAPI (4’,6-diamidino-2-phenylindole) and excluded from analysis where appropriate. Primary antibodies specific for the following markers were directly conjugated to FITC, PE, APC, PerCP-Cy5.5, eFluor-780, eFluor-450, or PE-Cy7 and purchased from eBioscience (CD3, CD4, CD8α, CD11b, CD16/32, CD34, CD45.1, CD45.2, CD45R(B220), CD49b(DX5), FcεR1 and Gr1) or from BD Biosciences (c-kit, IL-5Rα, Siglec F, CCR3, and Annexin V). For intracellular staining, surface-labeled cells were fixed in isotonic 3% paraformaldehyde and incubated with affinity-purified anti-CF (Hamilton et al., 2008 (link)) in Fix & Perm (Invitrogen) according to product instructions. Staining was detected with highly cross-adsorbed donkey anti-rabbit-FITC (Jackson Immunochemicals, Stratech, UK). For cell sorting experiments, eosinophil populations were sorted on a BD Influx flow cytometer.
Cd16 32
Manufactured by BD
Sourced in United States
The CD16/32 is a laboratory equipment used for the analysis of cell surface markers. It is a flow cytometry reagent that can be used to identify and quantify cells expressing the CD16 and CD32 antigens.
Automatically generated - may contain errors
Lab products found in correlation
Cd11b, by BD (8 mentions)
Lsm 710, by Zeiss (6 mentions)
F4 80, by BD (5 mentions)
Cd11c, by BD (4 mentions)
Siglec f, by BD (3 mentions)
F4 80, by BioLegend (3 mentions)
Cd206, by BD (3 mentions)
Il 5rα, by BD (2 mentions)
Dispase, by BD (2 mentions)
Facsaria 2 machine, by BD (2 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (2 mentions)
Mhcii, by BD (2 mentions)
Cd206, by R&D Systems (2 mentions)
Percoll, by Merck Group (2 mentions)
Cd11b, by BioLegend (2 mentions)
Facsaria, by BD (2 mentions)
Cd11b, by Thermo Fisher Scientific (2 mentions)
Ack lysing buffer, by Thermo Fisher Scientific (2 mentions)
Neuronal nuclei (neun), by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
49 protocols using cd16 32
1
Analytical Flow Cytometry of Immune Cells
2
Isolation of Alveolar Type 2 Cells
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AT2 cells were isolated as previously described44 . Briefly, lungs were perfused with 0.9% saline, followed by inflation with 2.5 ml dispase (BD Biosciences 354235). Single cell suspensions were prepared in C-tubes (Miltenyi Biotec 130-096-334) using the gentleMACS dissociator (Miltenyi Biotec) with 120 U/ml DNaseI (Sigma D4527). Suspensions were filtered through 40 μm strainers, resuspended in MACS buffer (1X PBS + 2 mM EDTA + 0.05% BSA) and incubated with the following anti-mouse biotinylated antibodies: CD45 (Biolegend 103104, clone 30-F11), CD16/32 (BD Pharmingen 553143, clone 2.462), CD31 (Biolegend 102503, clone MEC13.3), CD90.2 (Biolegend 105304, clone 30-H12) and Ter119 (Biolegend 116203, clone Ter119). Suspensions were subsequently incubated with anti-biotin microbeads (Miltenyi Biotec 130-097-046) and purified over a LS column (Miltenyi Biotec 130-042-401) attached to a QuadroMACS separator (Miltenyi Biotec). Eluates from the column (CD45− CD16/32− CD31− CD90− Ter119−) were stored at −80 °C as a dry pellet for Western blot analysis or processed immediately for RNA isolation.
3
Quantification of RAW264.7 Cell Phenotypes
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RAW264.7 (1 × 104) cells cultured with HA and β‐TCP samples for 2 and 5 days were collected and then respectively incubated with peridinin chlorophyll protein (Percp, BioLegend, USA)‐conjugated anti‐mouse CD11c and activated protein C (APC, eBioscience, USA)‐conjugated anti‐mouse CD206 at room temperature for 30 min without light after fixation, membrane breaking and blocked with CD16/32 (BD pharmingen) steps. Finally, the cells were resuspended in 200 µL PBS and subjected to FACSCalibur flow cytometry (Beckman, CytoFLEXS, USA).
4
Flow Cytometry Cell Sorting
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BD FACS Aria II machine was used to phenotypically assess and sort the cells. Antibodies used to block Fc receptors: CD16/32 (564220, BD Pharmingen). Antibodies used to exclude nonviable cells from flow cytometry: Fixable Viability Dye eFluor 780 (65-0865-14, Affymetrix eBioscience).
5
Macrophage Phenotyping by Flow Cytometry
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Samples were incubated with an Fc receptor blocker (CD16/32, BD Bioscience) to reduce nonspecific antibody binding. Freshly isolated samples were resuspended in staining buffer (R&D Systems) and stained with F4/80‐FITC (eBioscience) for 30 min at 4 °C. For intracellular staining, we used the Intracellular Fixation and Permeabilization Kit (BD Bioscience); this was used in accordance with the manufacturer’s instructions. Cells were then washed and stained with iNOS-APC (eBioscience) and CD206-PE (eBioscience). Flow cytometry was performed using a FACS Aria flow cytometer (BD Bioscience) and data were analyzed with FlowJo V10.6.2 software (TreeStar, Ashland, OR).
6
SARS-CoV-2 RBD-Specific T Cell Response
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Mouse spleens were
harvested on D105 after vaccination, and single-cell suspensions were
prepared by processing them through a 70 μm cell strainer (BD
Biosciences). Cells were then incubated in FACS tubes at 6 ×
105 cells per tube and stimulated for 18 h with 2 μg/mL
RBD peptide mix [PepMix SARS-CoV-2 (S-RBD) Protein ID: P0DTC2 PM-WCPV-S-RBD-1,
JPT] or media alone. As a positive control, cells were stimulated
with antimouse CD3 [0.05 μg/mL] (Southern Biotech) and antimouse
CD28 antibody [0.5 μg/mL] (Southern Biotech). GolgiStop (BD
Biosciences) was added for the last 10 h of the assay. Following stimulation,
cells were washed, stained with Aqua live/dead viability dye (Thermo
Fisher) in PBS, washed two additional times, and stained with a cocktail
of monoclonal antibodies and Fc block: CD16/32, CD4 Ax700 RM4-5, CD8
APC-H7 53-6.7, and CD44 APC IM7 (all BD Bioscience). Cells were fixed
and permeabilized according to the manufacturer’s protocol
(BD Biosciences) and stained for intracellular cytokines with IFNγ
PE-Cy7 XMG1.2, TNFα BV650 MP6-XT22, and IL-4 BV786 11B11 (BD
Bioscience). Cells were washed, fixed with 2% formaldehyde, acquired
on a Cytek Aurora (Northern Lights), and analyzed using Cytobank V7.3.0.
harvested on D105 after vaccination, and single-cell suspensions were
prepared by processing them through a 70 μm cell strainer (BD
Biosciences). Cells were then incubated in FACS tubes at 6 ×
105 cells per tube and stimulated for 18 h with 2 μg/mL
RBD peptide mix [PepMix SARS-CoV-2 (S-RBD) Protein ID: P0DTC2 PM-WCPV-S-RBD-1,
JPT] or media alone. As a positive control, cells were stimulated
with antimouse CD3 [0.05 μg/mL] (Southern Biotech) and antimouse
CD28 antibody [0.5 μg/mL] (Southern Biotech). GolgiStop (BD
Biosciences) was added for the last 10 h of the assay. Following stimulation,
cells were washed, stained with Aqua live/dead viability dye (Thermo
Fisher) in PBS, washed two additional times, and stained with a cocktail
of monoclonal antibodies and Fc block: CD16/32, CD4 Ax700 RM4-5, CD8
APC-H7 53-6.7, and CD44 APC IM7 (all BD Bioscience). Cells were fixed
and permeabilized according to the manufacturer’s protocol
(BD Biosciences) and stained for intracellular cytokines with IFNγ
PE-Cy7 XMG1.2, TNFα BV650 MP6-XT22, and IL-4 BV786 11B11 (BD
Bioscience). Cells were washed, fixed with 2% formaldehyde, acquired
on a Cytek Aurora (Northern Lights), and analyzed using Cytobank V7.3.0.
7
Adoptive Transfer of Naive CD4+ T Cells
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Naive CD4+ T cells from B6/SJL mice expressing CD45.1 were negatively selected using a cocktail of biotin-conjugated antibodies (anti-CD11c, CD11b, B220, MHC-II, CD8, GR1, CD16/32 (BD Bioscience) and anti-CD25, CD44 (Biolegend) followed by separation with Streptavidin-microbeads (Miltenyi Biotec). 6x106 cells were adoptively transferred into recipient mice. IL-17 and IL-22 intracellular production by flow cytometry in PPs T cells was determined 14 days after transfer by PMA and Ionomycin re-stimulation and intracellular cytokine staining followed by flow cytometry.
8
Myelination and Microglia Analysis in Tissue Sections
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Paraffin sections (4 μm) were used for MBP (1:200, Millipore, AB980) and dMBP (1:2000, Millipore, AB5864) staining to determine the degree of myelination and the production of degraded myelin debris respectively. Frozen sections (25 μm) were prepared for staining with Iba1 (1:400, Wako, Japan), Arg1 (1:200, Santa Cruz, USA), iNOS (1:200, Santa Cruz, USA), CD206 (1:100, R&D, USA), CD16/32 (1:500, BD, USA), and O4 (1:50, Millipore, USA) to determine the recruitment of microglia into the area of CC and the phenotype of the recruited microglia. The sections were first washed three times and blocked with 4% goat serum in 0.3% Triton X-100 for 3 h at room temperature followed by incubation with antibodies described above at 4°C for 12 h followed by Alexa Fluor 488 or 555 goat anti-rabbit secondary antibodies (1:1000, Invitrogen, USA) at 37°C. All sections were treated with Fluorescence Decay Resistant Medium with DAPI before being covered with coverslip. The images were captured by a multiphoton laser scanning confocal microscopy system (Olympus Fluoview FV1000) or common fluorescence microscope (Leica DMI6000).
9
Liver Macrophage Isolation Protocol
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Whole naïve or granulomatous livers were chopped into fine pieces with a razor blade and digested in 100 units/ml collagenase (Sigma) for 1 hr at 37oC with rocking. The tissue was then ground into a single-cell suspension through a 100-µm nylon mesh. Hepatocytes were pelleted out with a 50 g spin for 5 min for cleaner density separation. Leukocytes were separated on a 40% Percoll (Sigma-Aldrich) gradient (2000 rpm for 15 min) and treated for 2 min with 1 ml ACK (ammonium chloride–potassium bicarbonate) lysis buffer to lyse erythrocytes. Leukocytes were stained for 30 minutes with antibodies for CD16/32 (BDBiosciences), CD45 (Biolegend; San Diego, CA), CD11b (Biolegend), Siglec F (BDBiosciences), Ly6G (BDBiosciences), F4/80 (Biolegend), CD64 (Biolegend), and Ly6C (Biolegend) diluted in FACS buffer. CD45+ SiglecF- CD11b+ Ly6G- F4/80+ CD64+ cells were sorted with at least 90% purity from amongst the stained cells using a FACS Aria (BDBiosciences).
10
Intraperitoneal Thioglycollate-Induced Macrophage Activation
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Mice were intraperitoneally (i.p.) injected with 2 ml 3% thioglycollate (BD; Franklin Lakes, NJ) to elicit peritoneal macrophage recruitment or left untreated, as indicated. 4 days later, peritoneal cells from both groups were harvested as described above, and equal numbers of cells per mouse were resuspended with 20 ng/ml recombinant murine IL-4 (Peprotech) in complete RMPI or complete RMPI alone. The resuspended cells were placed in a 37°C water bath for 30 minutes with periodic agitation. Next, cells were fixed with 1.5% paraformaldehyde, washed, and permeabilized with cold methanol overnight at −20°C. Permeabilized cells were washed twice with PBS containing 0.1% bovine serum albumin and stained with anti-mouse STAT6 (BD), F4/80 (Biolegend), CD11b (eBioscience), Gr1 (BD Pharmingen), and CD16/32 (BD) for 1 hour on a shaker at room temperature. Phosphorylation of STAT6 in F4/80hiCD11bhiGr1– macrophages and F4/80l°CD11bloGr1– lymphocytes was measured using a BD FACSCanto II flow cytometer and FlowJo v.7.6 software (Tree Star; Ashland, OR).
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