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Rat anti mouse cd16 32 antibody

Manufactured by BD
Sourced in United States

The Rat anti-mouse CD16/32 antibody is a lab equipment product used for the detection and analysis of the CD16 and CD32 receptors in mouse samples. It is a primary antibody that binds to these specific cell surface markers, allowing researchers to identify and study cells expressing these proteins.

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18 protocols using rat anti mouse cd16 32 antibody

1

Phosphorylated p38 MAPK Activation in Neutrophils

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Cell staining was performed in the presence of purified rat anti-mouse CD16/32 antibody (BD). Antibodies used for staining were as follows; anti-Ly6G-PE (1A8, BD), anti-CD11b-APC (M1/70, eBioscience), anti-Ly6G-APC-Cy7 (1A8, BD), anti-Ly6C-APC-Cy7 (AL-21, BD), mouse biotin-conjugated anti-CD45 (30-F11, eBioscience), anti-streptavidin-V500 (BD), and anti-7-AAD (BioLegend).
For intracellular staining for phosphorylated p38 MAPK, 1 × 106/ml of bone marrow-derived neutrophils were stimulated with 1 μg/ml of R848 (Enzo Life Sciences) for 30 min in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS), 100 μg/ml of L-glutamine (Sigma-Aldrich), 100 U/ml of penicillin (Sigma-Aldrich), 100 μg/ml of streptomycin (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Stimulated neutrophils were stained with anti-CD11b-APC (M1/70, eBioscience) and anti-Ly6G-APC-Cy7 (1A8, BD) antibodies in the presence of purified rat anti-mouse CD16/32 antibody (BD), fixed with Lyse/Fix Buffer (BD), and permeabilized by BD Phosflow Perm Buffer II (BD). Then, neutrophils were intracellularly stained with Alexa Fluor 488 Mouse IgG1κ isotype control (BD) or Alexa Fluor 488 mouse anti-p38 MAPK (pT180/pY182) (BD).
Flow cytometric analysis was performed using MoFlo XDP and data were analyzed using FlowJo Software (Tree star).
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2

Comprehensive Tumor Immunophenotyping via Flow Cytometry

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For analysis of tumors and lymphoid organs derived from the MOC1 syngeneic model, antibodies specific for mouse CD3 (560590; 17A2), CD4 (740024; H129.19), CD8a (55795; 53-6.7), CD44 (560780; IM7), CD62L (562404; MEL-14), CD69 (552879; H1.2F3), CD25 (562694; PC61), Foxp3 (560403; MF23), T-bet (561264; 4B10), and CXCR5 (560617; 2G8), were purchased from BD Biosciences, California, USA. Intracellular staining of transcription factors was performed using Fixation/Permeabilization solution kit (554714) or Mouse Foxp3 Buffer set (560409) from BD Biosciences. All samples were stained after Fc blocking using 5ul/test of purified rat anti–mouse CD16/32 antibody (BD Biosciences). Samples were acquired using FACS Aria II cell sorter (BD Biosciences, California, USA) and data analysis was performed using Flowjo® software v10 (Flowjo LLC, BD Biosciences, California, USA). MFI is presented as the median value of the samples.
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3

Antigen-Specific T Cell Proliferation

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Splenocytes were isolated from mice at day 8 following i.p. infection with 2.5 × 105 PFU of the DM strain of mouse hepatitis virus (MHV-DM). Enriched populations of CD4+ and CD8+ T cells, isolated according to the manufacturer’s instructions (CD4 and CD8 Isolation kits, Miltenyi Biotec, Auburn, CA, USA), were labeled with the fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Grand Island, NY, USA), at 2.5 μM final concentration. Then 1 × 106 total cells per well were incubated with FTY720P 100 nM or vehicle and stimulated with 5 μM final peptide concentration of CD4+ T cell immunodominant epitope M133-147, CD8+ T cell immunodominant epitope S510-518, or non-specific OVA control, and cultured for 72 h at 37°C, 5% CO2 in complete media. Cells were then washed and the Fc receptor blocked with 1 × PBS containing 1% BSA and a 1:200 dilution of rat anti-mouse CD16/32 antibody (Pharmingen, San Jose, CA, USA). Next, cells were stained for surface antigens using APC-conjugated rat anti-mouse CD4 and CD8 (Pharmingen, San Jose, CA, USA), according to the viral peptide stimulation condition, for 45 min at 4°C. Cells were analyzed and the data assessed as described above.
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4

Characterization of Thymocyte Subsets

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Total thymocytes were isolated from the thymus in HSC buffer (HBSS with Ca2+ and Mg2+, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ACK lysing buffer (Lonza). 1 x 106 live thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with a combination of the antibodies including PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5, eBioscience), PE conjugated anti-mouse CD8 (clone: 53-6.7, eBioscience), APC conjugated anti-mouse CD44 (clone: IM7, eBioscience), BV421 conjugated anti-mouse CD25 antibodies (clone: PC61, BioLegend). FITC conjugated anti-mouse CD45.2 (clone: 104, eBioscience), and APC-eFluor780 conjugated anti-mouse CD45.1 (clone: A20, e eBioscience). All antibodies were diluted 1:400. Data were collected from at least 100,000 single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (C-LL).
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5

Thymocyte Isolation and Analysis

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Total thymocytes were isolated from the thymus in haematopoietic stem cell (HSC) buffer (Hank's Balanced Salt Solution with Ca2+ and Mg2+, 5% fetal bovine serum, 2 mM EDTA). Red blood cells (RBC) were lysed using ammonium-chloride-potassium (ACK) lysing buffer (Lonza). Total number of thymocytes was counted with a Coulter counter (Beckman Coulter). 1 × 106 thymocytes were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with phycoerythrin (PE)-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE-conjugated anti-mouse CD8 (clone: 53-6.7), allophycocyanin (APC) conjugated anti-mouse CD44 (clone: IM7), fluorescein isothiocyanate (FITC) conjugated CD25 (clone: PC61.5) and APC-eFluor780 conjugated anti-mouse cKit (clone: 2B8) antibodies (eBioscience). All antibodies were diluted 1:400. Dead cells were excluded by staining with Calcein Blue AM (Life technologies). Data were collected from 250,000 single cells by FACSCanto (BD Pharmingen) and analysed by FlowJo (Tree Star, Inc.) without knowledge of the genotype or treatment by a single observer (C.L.L.).
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6

Multiparametric Flow Cytometry Analysis of Murine Hematopoietic Cells

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Whole bone marrow (WBM) cells were isolated from femurs and tibias by grinding the bones in HSC buffer. RBCs were lysed using ACK lysing buffer (Lonza). Total number of WBM cells was counted with a Coulter counter (Beckman Coulter). Three million WBM cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated anti-mouse CD3e (clone: 145-2C11), PE-Cy5 conjugated anti-mouse CD4 (clone: GK1.5), PE-Cy5 conjugated anti-mouse CD8 (clone: 53-6.7) PE-Cy5 conjugated anti-mouse B220 (clone: RA3-6B2), PE-Cy5 conjugated PE-Cy5 conjugated anti-mouse CD11b (clone: M1/70), PE-Cy5 conjugated anti-mouse Gr-1 (clone: RB6-8C5), PE-Cy5 conjugated anti-mouse Ter119 (Ly-76), PE conjugated anti-mouse Sca1 (clone:D7), APC conjugated anti-mouse cKit (clone: 2B8), FITC conjugated anti-mouse CD48 (clone: HM48-1) (eBioscience) and Brilliant Violet 421 conjugated anti-mouse CD150 (clone: TC15-12F12.2) antibodies (BioLegend). All antibodies were diluted 1:400. Dead cells were excluded by staining with 7-AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Pharmingen) and analyzed by FlowJo (Tree Star, Inc) without knowledge of the genotype or treatment by a single observer (CLL).
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7

Retinal Flow Cytometry Analysis

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Retinal flow cytometry was performed as previously described (60 (link)). Retinas were collected on P15 or P17 and then digested with papain (1.65 U/mL; Worthington Biochemical) by incubation in a 37°C water bath for 16 minutes with a gentle swirl of the tube every 8 minutes. Tissues were then disaggregated by gentle pipetting up and down, and dropwise passing back into the tube through a serological pipette in low-ovomucoid solution. Single-cell suspensions of tissues were obtained by passing through a 70 mm cell strainer (BD Biosciences). The cells were then washed twice with PBS containing 5% FBS, 0.05% NaN3, and HEPES (10 mM) and blocked with rat anti-mouse CD16/32 antibody (BD Biosciences) for 5 minutes at 4°C. Cells were incubated with rat anti-mouse APC-CD11b (eBioscience) and FITC-CD45 (eBioscience) for 30 minutes at room temperature. Cells were washed 3 times. A 4-laser Sony SH800 cell sorter (Sony Biotechnology) was used to collect the data, and FlowJo software was used for analysis. Antibodies used for staining are listed in Supplemental Table 1.
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8

Isolation and Analysis of Mouse Hematopoietic Stem Cells

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Whole bone marrow cells were isolated from femurs and tibias by grinding the bones in hematopoietic stem cell (HSC) buffer [Hanks’ balanced salt solution (HBSS) with Ca2+ and Mg2+, 5% fetal bovine serum, 2 mM EDTA]. Red blood cells (RBCs) were lysed using ACK lysing buffer (Lonza, Basel, Switzerland). The total cell number was counted with a Coulter counter (Beckman Coulter Inc., Brea, CA). Three million cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail (anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies), PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (eBioscience). Dead cells were excluded by staining with 7-AAD (BD Pharmigen). Data were collected from 1 million single cells by FACSCanto (BD Biosciences) and analyzed by FlowJo software.
For colony-forming cell (CFC) assays, 2 × 104 RBC-depleted whole bone marrow cells were plated in MethoCult GF 3434 (StemCell Technologies) and colonies were counted 7 days later.
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9

Isolation and Analysis of Regulatory B Cells

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Splenocytes were isolated using a 40-µm nylon cell strainer (BD Biosciences, San Jose, CA, USA); RBCs were lysed with buffer containing 0.14 NH4Cl and 0.017 M Tris-base (pH 7.5). IL-10-producing CD1dhighCD5+CD19+ Bregs were analyzed by flow cytometry after immunostaining of surface markers with the following monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-CD19 (clone 1D3) (BD Biosciences, San Jose, CA, USA), phycoerythrin (PE)-conjugated anti-CD1d (clone 1B1; isotype, Rat IgG2b, κ) (BD Biosciences), allophycocyanin (APC)-conjugated anti-CD5 (clone 53–7.3) (eBioscience, San Diego, CA, USA), and peridinin chlorophyll-protein complex (PerCP)- or PE-conjugated anti-IL-10 (clone JES5–16E3) (eBioscience). For subsequent intracellular IL-10 staining, the Cytofix/Cytoperm kit (BD Biosciences) was used according to the manufacturer's protocol (BD Biosciences). Intracellular transport processes were inhibited with BD GolgiStop containing the transport inhibitor monesin and Fc receptors were blocked by treatment with rat anti-mouse CD16/32 antibody (BD Bioscience) for 15 min. The FlowJo software (Tree Star Inc., Ashland, OR, USA)was used to analyze the flow cytometry data. To determine background staining, non-reactive isotype-matched control monoclonal antibodies (eBioscience) were used and cells were gated to exclude ≥98% of non-reactive cells.
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10

Murine Hematopoietic Stem Cell Isolation

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Whole bone marrow cells were isolated from femurs and tibias by grinding the bones in hematopoietic stem cell (HSC) buffer [Hanks’ balanced salt solution (HBSS) with Ca2+ and Mg2+, 5% fetal bovine serum, 2 mM EDTA]. Red blood cells (RBCs) were lysed using ACK lysing buffer (Lonza, Basel, Switzerland). Cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail containing anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies (eBioscience). Cells were stained with either PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (eBioscience) or with CD27 conjugated to APC (Thermo Fisher) and CD201 conjugated to PE (eBioscience). Dead cells were excluded by staining with 7-AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Biosciences) and analyzed by FlowJo software.
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