Fcγ receptors were blocked by preincubation with saturating amounts of anti-mouse CD16/32 (BD Pharmingen) or normal mouse serum. All stainings were performed in PBS containing 2% heat inactivated new born calf serum at 4°C, except for the CCR7 staining which was performed at 37 °C. For intracellular Foxp3, IRF4 and IRF8 staining, cells were stained for extracellular markers, fixed and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen) prior to intracellular staining. Stained cells were analyzed using FACSCanto™ II (BD biosciences) and FlowJo software.
Anti mouse cd16 32
Manufactured by BD
Sourced in United States
Anti-mouse CD16/32 is a laboratory reagent used to detect the expression of the CD16 and CD32 receptors on mouse cells. It is a tool for immunological research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
7 aad, by BD (3 mentions)
Lsrii flow cytometer, by BD (3 mentions)
Facsdiva software, by BD (3 mentions)
Cytofix cytoperm solution, by BD (2 mentions)
Dnase 1, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Recombinant mouse il 3, by Thermo Fisher Scientific (2 mentions)
Recombinant mouse il 33, by R&D Systems (2 mentions)
Pe conjugated anti mouse c kit ab, by BD (2 mentions)
Apc conjugated anti mouse st2 ab, by BD (2 mentions)
Dnp bsa, by Cosmo Bio (2 mentions)
Evans blue, by Merck Group (2 mentions)
Cd11b, by BD (2 mentions)
F4 80, by BD (2 mentions)
Cd138, by BioLegend (2 mentions)
Live dead dye, by Thermo Fisher Scientific (2 mentions)
Mhcii, by BioLegend (2 mentions)
Lsrfortessa cell analyzer, by BD (2 mentions)
Fitc conjugated anti ly6g, by BD (2 mentions)
45 protocols using anti mouse cd16 32
1
Blocking Fcγ Receptors for Cellular Analysis
2
Isolation and Quantification of Murine MSCs
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In the experimental group, BMCs were obtained from murine femurs and tibias 1 week after preparation of periodontal defects. Untreated mice were used as controls. To evaluate the ratio of bone marrow MSCs for all collected cell counts, we conducted at least three experiments using two mice in the experimental and control groups. BMCs were obtained as described previously (Morikawa et al., 2009 (link)). Briefly, femurs and tibias were aseptically removed from two mice and bones were crushed with an ice-cold pestle and mortar. Bone chips, including marrow were rinsed with HBSS+ and were digested using collagenase (#032-22364; Wako) for an hour at 37°C. Collected BMCs were hemolyzed and FcR was blocked with anti-mouse CD16/32 (#553142; BD) on ice for 5 min. BMCs (2–5 × 105) were multi-stained with CD45.2-APC-eFlour780 (#47-0454; eBioscience, San Diego, CA, USA), TER119-PECy7 (#25-5921; eBioscience), pdgfrα-PE (#12-1401-81; eBioscience), Sca-1-APC (#17-5981-81; eBioscience) (1: 100 dilution, 30 min, on ice) and 7AAD (#559925; BD) (1: 100 dilution, 10 min, on ice). The ratio of MSCs [CD45.2 (−), TER119 (−), 7AAD (−), pdgfrα (+), Sca-1 (+)] in BMCs was evaluated by FACS analysis.
3
Cytokine and Signaling Analysis in Infected Lung
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For cellular analysis, inflammatory cytokines measurement and signaling pathway analysis, further experimental infections as described above were performed, but the mice only received 100 kU/kg dosage of rhCAT.
On days 2 and 4 post-infection, the mice (n = 4, per group) were euthanized. Lungs were harvested from PBS-perfused mice and diced using surgical scissors. Diced tissue was suspended in 4 mL of CDTI buffer [0.5 mg/mL collagenase from Clostridium histolyticum type IV (Sigma), 50 U/mL Dnase I (Sigma), 1 mg/mL trypsin inhibitor type Ii-s (Sigma) in DMEM] for 1 h at 37 °C. The suspension was then passed through a 100-μm filter, and unwanted red blood cells were lysed using red blood cell lysis buffer [0.02 Tris–HCl (pH 7.4), 0.14 NH4Cl]. Inflammatory cells were purified by centrifugation in 35 % PBS-buffered Percoll (GE Healthcare Life Sciences) at 500 × g for 15 min. Cell pellets were resuspended in staining buffer (RPMI-1640 medium), and Fc receptors were blocked using 25-μg/mL anti-mouse CD16/32 (BD Biosciences). Cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD11b+, F480−, Ly6G+ (Neutrophils), CD11b+, F480+, and Ly6G− (Macrophage/monocytes).
On days 2 and 4 post-infection, the mice (n = 4, per group) were euthanized. Lungs were harvested from PBS-perfused mice and diced using surgical scissors. Diced tissue was suspended in 4 mL of CDTI buffer [0.5 mg/mL collagenase from Clostridium histolyticum type IV (Sigma), 50 U/mL Dnase I (Sigma), 1 mg/mL trypsin inhibitor type Ii-s (Sigma) in DMEM] for 1 h at 37 °C. The suspension was then passed through a 100-μm filter, and unwanted red blood cells were lysed using red blood cell lysis buffer [0.02 Tris–HCl (pH 7.4), 0.14 NH4Cl]. Inflammatory cells were purified by centrifugation in 35 % PBS-buffered Percoll (GE Healthcare Life Sciences) at 500 × g for 15 min. Cell pellets were resuspended in staining buffer (RPMI-1640 medium), and Fc receptors were blocked using 25-μg/mL anti-mouse CD16/32 (BD Biosciences). Cells were stained with fluorescently labeled antibodies against the following mouse proteins: CD11b+, F480−, Ly6G+ (Neutrophils), CD11b+, F480+, and Ly6G− (Macrophage/monocytes).
4
Single Cell Immune Profiling Protocol
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Single cell suspensions from the spleen and liver were acquired according to the methods previously described (21 (link)) and analyzed using flow cytometry. Antibodies used for flow cytometry staining including Percp-Cy5.5-anti-mouse-CD45.1, APC-Cy7-anti-mouse-CD11b, Percp-Cy5.5-anti-mouse-NK1.1, BV650-anti-mouse-H2-Kb were purchased from BD Bioscience (SanDiego, CA, USA); purified anti-mouse-CD16/32, APC-anti-mouseCD43, PE-anti-mouse-NKp46, APC-anti-mouse-CD107, FITC-anti-mouse-NKG2D, PE-anti-mouse-IFN-γ, PE/Cy7-anti-mouse-TNF-α, FITC-anti-mouse-CD62E, PE-anti-mouse-CD4, PE/Cy7-anti-mouse-CD44, APC/Cy7-anti-mouse-CD62L, Pacific Blue-anti-mouse-CD8a, FITC-anti-mouse-CD69 were purchased from Biolegend (San Diego, CA, USA); PE-Cy7-anti-mouse-Granzyme B, APC-anti-mouse-Perforin were purchased from eBioscience (San Diego, CA, USA). Recombinant Mouse E-Selectin, P-Selectin, and L-Selectin chimera were purchased from Biolegend (San Diego, CA, USA) for detecting the binding abilities. Samples were detected on a NovoCyte Flow Cytometer (ACEA Biosciences, San Diego, CA, USA) and data were analyzed by using Flowjo software (Flowjo, Ashland, OR, USA).
5
Cytokine profiling of stimulated immune cells
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Cultured or freshly isolated cells from spleen were stimulated with PMA (500 ng/ml) and ionomycin (50 ng/ml; both from Sigma–Aldrich) for 4 h; GolgiStop was added during the final 3 h. The cells were incubated with anti-mouse CD16/32 to block non-specific Fc binding (BD Biosciences) followed separately by PerCP-conjugated anti-CD3, anti-CD4, (BD Biosciences) or appropriate isotype controls. Cells were then fixed with Cytofix/Cytoperm buffer (BD Biosciences), permeabilized with perm/wash buffer (BD Biosciences), and incubated with FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-4 (all from BD Biosciences) or isotype controls followed by incubation with secondary antibodies or streptavidin if necessary. The cells were analysed on dual laser (488 nm & 633 nm) FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest Pro software (Becton Dickinson, Mountain View, CA).
6
Quantification of Tumor Immune Cells by Flow Cytometry
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The content of inflammatory cells was quantified by flow cytometry as described
[58 (link)]. Briefly, tumor tissues were cut into multiple small cubes and digested in an enzyme mixture for 45 mins at 37°C. The cell suspension was centrifuged and pre-incubated with Fc-γ block antibody (anti-mouse CD16/32; Pharmingen, San Diego, CA, USA) to prevent nonspecific binding. Cell staining involved different combinations of fluorochrome-coupled antibodies to CD45, F4/80, CD206 for 30 mins at 4°C in the dark. Fluorescence data were collected by use of an EPICS XL flow cytometer (Beckman Coulter) and analyzed by use of Cellquest (Beckman). Fluorescence minus one (FMO) controls were included to determine the level of nonspecific staining and auto-fluorescence associated with subsets of cells in each fluorescence channel.
[58 (link)]. Briefly, tumor tissues were cut into multiple small cubes and digested in an enzyme mixture for 45 mins at 37°C. The cell suspension was centrifuged and pre-incubated with Fc-γ block antibody (anti-mouse CD16/32; Pharmingen, San Diego, CA, USA) to prevent nonspecific binding. Cell staining involved different combinations of fluorochrome-coupled antibodies to CD45, F4/80, CD206 for 30 mins at 4°C in the dark. Fluorescence data were collected by use of an EPICS XL flow cytometer (Beckman Coulter) and analyzed by use of Cellquest (Beckman). Fluorescence minus one (FMO) controls were included to determine the level of nonspecific staining and auto-fluorescence associated with subsets of cells in each fluorescence channel.
7
Identification of Liver Immune Cells
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Single cells were isolated from fresh liver and tumor tissues by gentle MACS Dissociators (Miltenyi Biotec). Fc receptors were blocked by antimouse CD16/32 (BD Pharmingen), and cells were stained with antimouse surface marker antibodies (online supplemental table S2 ). Flow cytometry was performed on FACSAria Fusion platform (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). The expression of surface markers was detected to identify different immune cell subsets (online supplemental table S3 ). Absolute numbers were calculated by multiplying frequencies obtained from flow cytometry with the total live mononuclear cell count, and then divided by liver weight. For identification of different macrophage phenotypes, surface markers were referred to online supplemental table S4 .
8
Immunophenotyping Cells by Flow Cytometry
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In general, 106 cells for surface only stains or 2x106 cells for stains investigating intracellular antigens were stained in round bottom 96 well plates. Surface antibodies were diluted with staining buffer (1% FBS, 1 mM EDTA, and 0.02% NaN3 in PBS) into cocktails containing Fc block (purified anti-mouse CD16/32, BD Pharmingen, San Diego, CA) and added to cells at 45 μl per sample. Cells were washed and resuspended in staining buffer for analysis within 24 h. Data were collected using a BD Fortessa instrument running FACS DIVA software. Data were analyzed using FlowJo v10 (TreeStar, Ashland, OR).
9
Mast Cell Activation Signaling
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Reagents used in this study were acquired from the indicated suppliers: SR9009 (Merck Millipore, Burlington, MA, USA); SR9011, GSK4112, LPS, and Evans blue (Sigma-Aldrich, St. Louis, MO, USA); recombinant mouse IL-3 (PeproTech, Rocky Hill, NJ, USA); recombinant mouse IL-33 (R & D Systems, Minneapolis, MN, USA); anti-TNP IgE, anti-DNP mouse IgE mAb, anti-mouse CD16/32, PE-conjugated anti-mouse c-kit Ab, and APC-conjugated anti-mouse ST2 Ab (BD Bioscience, San Jose, CA, USA); DNP-BSA (Cosmo Bio, Tokyo, Japan); APC-conjugated anti-mouse CD63 Ab, FITC-conjugated anti-mouse FcεRIα (BioLegend, San Diego, CA, USA); anti-phospho-NF-κB p65 Ab (Ser536; #3033), anti-phospho-p38 MAPK Ab (Thr180/Thy182; #4511), anti-phospho-Gab2 Ab (Tyr452; #3882), anti-phosph-PI3 Kinase p85 (Tyr458)/p55 (Tur199) Ab (#4228), and anti-β-actin Ab (#4970) (Cell Signaling Technology, Danvers, MA, USA).
10
Isolation and Analysis of Immune Cells
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Animals were euthanized via inhalation of a lethal dose of isofluorane and cardiac perfusion with PBS was performed. Spinal cords or cervical lymph nodes were dissected into single-cell suspension, depleted of red blood cells via ammonium-chloride-potassium, and passed through a discontinuous Percoll gradient as previously described [19 (link)]. Single-cell suspensions were filtered, washed, and counted before being blocked with anti-mouse CD16/32 (1:200; BD Biosciences). Cells were subsequently stained with anti-CD4 (FITC-conjugated GK1.5; BD Biosciences), anti-CD8 (PE-Cy7-conugated Ly-2; BD Biosciences), anti-CD45 (APC-conjugated 30-F11; eBioscience), anti-F4/80 (FITC-conjugated Ci-A3-1; Serotech), anti-FOXP3 (EF660-conjugated FJK-16s; eBioscience) or PE-conjugated tetramers I-Ab/M133–147 and Db/S510–518 (8 μg/ml; National Institute of Allergy and Infectious Diseases MHC Tetramer Core Facility). Single-stain samples and IgG or IgM isotype controls were used to establish PMT voltages, gating, and compensation parameters. Cells were processed using an LSR II flow cytometer (BD) and analyzed using FlowJo software (Tree Star).
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