Cells were stained according to standard protocols. Cells were resuspended in PBS and treated with 1% ammonium chloride solution (10 min on ice). 1×105 cells/FACS tube were resuspended in 100 μl of PBS and incubated on ice for 10 min with a purified rat anti-mouse CD16/32 antibody (BioLegend, Fell, Germany) to block non-specific antibody binding. After blocking, cells were stained for 20 min on ice. The following antibodies were used for flow cytometric analyses of murine bone marrow cells and permanently growing cells of a diseased mouse: Ter119 and CD36 labeled with APC (both BioLegend), B220 and Ter119 labeled with eFluor450 (both eBioscience, Frankfurt, Germany), Mac-1 labeled with qDot605 (BioLegend). Antibodies labeled with Alexa Fluor 700 were used against the epitope of c-kit (eBioscience), Gr-1 was labeled with APC-Cy7 (both BioLegend), Sca-1 with PE-Cy5.5 (Caltag) and CD71 with PE-Cy7 (Becton Dickinson and BioLegend, respectively). Cells were analyzed using a FACS FORTESSA LSR II (Becton Dickinson, Heidelberg, Germany).
Rat anti mouse cd16 32 antibody
Manufactured by BioLegend
Sourced in Germany
The Rat anti-mouse CD16/32 antibody is a laboratory tool used for the detection and identification of the CD16/32 antigen, also known as the Fc gamma receptor III and II. This antibody can be utilized in various immunological techniques, such as flow cytometry, to study the expression and distribution of the CD16/32 antigen in mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Ter119, by BioLegend (1 mentions)
Anti cd45 percp, by BD (1 mentions)
Anti cd11c fitc, by BioLegend (1 mentions)
Anti cd31 pe cy7, by BioLegend (1 mentions)
Anti ly6g apc cy7, by BioLegend (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Anti cd326 apc, by Thermo Fisher Scientific (1 mentions)
Canto 2, by BD (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Fv 100d, by Olympus (1 mentions)
Rabbit anti mouse iba 1 antibody, by Fujifilm (1 mentions)
4 protocols using rat anti mouse cd16 32 antibody
1
Murine Bone Marrow Cell Immunophenotyping
2
Flow Cytometric Analysis of Lung Cells
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Isolated single lung cells were incubated with a rat anti-mouse CD16/32 antibody (Biolegend) for 30 min to block Fc receptors. Fluorophore-conjugated antibodies were added at the recommended dilutions, and the cells were then incubated with the specific antibodies in the dark for an additional 30 min. The following anti-mouse antibodies were used for cell surface marker staining: anti-CD31-PE/Cy7 (endothelial cell marker), anti-CD54-PE (a surrogate marker of antigen-presenting cell activation), anti-CD11c-FITC, and anti-Ly6G-APC/Cy7 (expressed on neutrophils) antibodies purchased from Biolegend (San Diego, CA); an anti-CD326-APC (epithelial cell surface marker) antibody purchased from eBioscience (San Diego, CA); and an anti-CD45-Percp (leukocyte common antigen) antibody purchased from BD Bioscience (San Jose, CA). Data acquisition and data analysis were performed with an Agilent NovoCyte flow cytometer and NovoExpress Software, respectively. We defined cell compartments as follows: endothelial, CD326− CD45−/CD31+; and neutrophil, CD45+/Ly6G+.
3
Analysis of PD-L1 Expression
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Cells were dislodged by scraping in 0.5 ml of PBS. After washing, samples were treated with rat anti-mouse CD16/32 antibody (1:1000, clone 93, BioLegend ref. 101302) at 4°C for 15 min to block Fc receptors. ~ 1 × 106 cells per sample were washed once and incubated with rat anti-mouse CD274/PD-L1 antibody (1:200, clone 10F.9G2, Biologend ref: 124308) for for 15 min at 4°C. After, cells were washed with 1 ml FACS buffer (PBS with 2% FCS). Samples were then incubated in 100 μl of fixative (eBiosciences FOXp3/Transcription factor staining buffer set, ref: 00-5523-00) for 10 minutes at room temperature. Cell were then washed in 1 ml permeabilization buffer (eBiosciences FOXp3/Transcription factor staining buffer set, ref: 00-5523-00), and stored in 100 μl of permeabilization buffer at 4°C. Right before acquisition, cells were washed once more in 1 ml of permeabilization buffer and resuspended in 100 μl of PBS containing 2.5 mM of EDTA. Flow cytometric analysis was performed using a Canto II (BD) instrument, acquiring a minimum of 1x105 singlets/sample. Results were exported as FCS files and analysed using FlowJo V10 (Tree Star) software.
4
Imaging Neuroinflammation and Neurodegeneration in Ischemic Brain
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Mice were sacrificed 3 days after ischaemia. The brains were removed following transcardial perfusion with 0.9% saline and 4% paraformaldehyde. Brains were postfixed in 4% paraformaldehyde overnight and then cryoprotected by immersion in 20% sucrose for 48 h. Coronal sections (30 μm) were prepared and immunostained as previously described18 (link). In brief, brain slices were incubated with rabbit anti-mouse Iba1 antibody (dilution 1:500; WAKO, Osaka, Japan), mouse anti-mouse NeuN antibody (dilution 1: 500; Millipore, Schwalbach, Germany), and rat anti-mouse CD16/32 antibody (dilution 1:300; Biolegend) overnight at 4 °C. Antibody binding was visualized with Alexa Fluor 488- and Alexa Fluor 594-labelled secondary antibodies (dilution 1:500; Invitrogen, Carlsbad, CA) using a laser confocal microscope (FV-100D; Olympus, Tokyo, Japan). Negative controls were prepared by omitting the primary antibodies and used to adjust confocal machine settings (i.e., gain and black level). The nuclei were stained with DAPI. Cells labelled with NeuN, or double-labelled with Iba1 and CD16/32, were counted in three representative micrographs of each hippocampal region (bregma −2.20 through −2.66) to quantify the fluorescent signal.
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