Total blood counts, leucocyte, and monocyte counts were measured with a Sysmex XN-450 Hematology Analyzer (Sysmex Corporation, Kobe, Japan). Undiluted blood (50 µL) was incubated for 15 min in the dark at room temperature with the following antibodies: CD3-APC-750 (mouse, dilution: 1:25; Beckman Coulter, Woerden, The Netherlands), CD56-APC (mouse, 1:25; Beckman Coulter), CD16-FITC (mouse, 1:25; eBioscience Thermo Fisher Scientific, Breda, The Netherlands), CD14-PC7 (mouse, 1:25; eBioscience Thermo Fisher Scientific), CCR2-BV421 (mouse, 1:50 and 1:25; BD Biosciences), CD36-APC-700 (mouse, 1:10; Beckman Coulter), CD41-PC5.5 (mouse, 1:50; Biolegend, San Diego, CA, USA), CD11b-BV785 (mouse, 1:50 and 1:25; Biolegend). Followed by the addition of 1 mL 10× diluted lysis buffer (BD Pharm Lyse; BD Biosciences), samples were mixed and incubated for 10 min in the dark at room temperature. Samples were then measured on a Beckman Coulter FC500 flow cytometer, each sample was measured both stained and unstained. Flow cytometry data were analysed using Kaluza software (Beckman Coulter); for gating strategy, see Supplementary material online , Figure S1 . Besides determining the different monocyte subsets (classical, intermediate, or non-classical). Atherogenic markers CCR2, CD36, CD41, and CD11b were determined per monocyte subset.
Ccr2 bv421
Manufactured by BD
The CCR2-BV421 is a fluorescently-labeled antibody that binds to the CCR2 receptor. CCR2 is a chemokine receptor expressed on the surface of various immune cells, including monocytes and macrophages. This antibody can be used to detect and quantify CCR2-expressing cells in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Cd56 apc, by Beckman Coulter (2 mentions)
Cd3 apc 750, by Beckman Coulter (2 mentions)
Cd36 apc 700, by Beckman Coulter (2 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Pharm lyse, by BD (2 mentions)
Cd11b bv785, by BioLegend (1 mentions)
Xn 450 hematology analyzer, by Sysmex (1 mentions)
Cd16 fitc, by BD (1 mentions)
Hla dr pe, by Beckman Coulter (1 mentions)
Cd14 pc7, by BD (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Cd45 ko, by Beckman Coulter (1 mentions)
Xn 450, by Sysmex (1 mentions)
Xn 9000, by Sysmex (1 mentions)
Cx3cr1 a488, by BioLegend (1 mentions)
Live dead violet dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cx3cr1 bv785, by BioLegend (1 mentions)
Mhcii a700, by BioLegend (1 mentions)
Cd11c bv785, by BioLegend (1 mentions)
Cd64 pe cy7, by BioLegend (1 mentions)
3 protocols using ccr2 bv421
1
Comprehensive Monocyte Phenotyping by Flow Cytometry
2
Immune cell profiling using flow cytometry
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Immune cell subset numbers were measured on a Sysmex XN-450 and Sysmex XN-9000 (Sysmex). FACS analysis was performed in one of the two participating study sites, because this method is sensitive to confounders when performed at different sites. A total of 50 μl of whole undiluted blood was incubated for a duration of 15 min in the dark at room temperature with the following antibodies: CD16-FITC (dilution 1:20), CD14-PC7 (1:20), CCR2-BV421 (1:20) (BD Biosciences, Vianen, the Netherlands); CD41-PC5.5 (1:20), CD11b-BV785 (1:20) (ITK Diagnostics BV, Uithoorn, the Netherlands); HLA-DR-PE (1:10), CD56-APC (1:10), CD3-APC-750 (1:10), CD45-KO (1:10), CD36-APC-700(1:10) (Beckman Coulter, Woerden, the Netherlands). Subsequently, 1 ml of lysis buffer (BD Pharm Lyse, BD Biosciences) was added, samples were mixed, incubated for another 10 min and then measured on a flow cytometer (Beckman Coulter FC500). To determine the position of analysis gates, single staining and fluorescence-minus-one control stains were used (Additional file 1 : Fig. S1). To analyse the FACS data, Kaluza software (Beckman Coulter, Woerden, the Netherlands) was used.
3
Multicolor Flow Cytometry of Immune Cells
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Flow cytometry data were acquired using a Cytek Aurora equipped with violet (405 nm), blue (488 nm), and red (640 nm) lasers. The following antibodies and stains were used: CCR2-BV421 (clone 475301, 747963; BD), Ly6C-BV510 (clone HK1.4, 128033; Biolegend), B220-BV605 (clone RA3-6B2, 103243; Biolegend), Ly6G-BV711 (clone 1A8, 127643; Biolegend), CD11c-BV785 (clone N418, 117336; Biolegend), CX3CR1-A488 (clone SA011F11, 149021; Biolegend), CX3CR1-BV785 (clone SA011F11, 149029; Biolegend), CD163-PE (clone TNKUPJ, 12-1631-80; Thermo Fisher Scientific), CD64-PE/Cy7 (clone X54-5/7.1, 139313; Biolegend), MRC1-PCP5.5 (clone C068C2, 141716; Biolegend), CD11b-PEFire640 (clone M1/70, 101279; Biolegend) TCRb-APC (clone H57-597, 109212; Biolegend), MHCII-A700 (clone M5/114.15.2, 107622; Biolegend), XCR1-APC/Cy7 (clone ZET, 148223; Biolegend), CD45-APC-Fire810 (clone 30F11, 103173; Biolegend), CD45.1-A647 (clone A20, 110720; Biolegend), CD45.2- BV785 (clone 104, 109839; Biolegend), and LIVE/DEAD Violet dead cell stain kit (L34955; Thermo Fisher Scientific). Flow cytometry data was analyzed using Flowjo 10 software. Dimensionality reduction and cluster identification were performed using the UMAP and Phenograph packages, respectively.
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