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Ccr2 fitc

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CCR2-FITC is a fluorochrome-conjugated antibody used for the identification and analysis of cells expressing the CCR2 chemokine receptor. It is a tool used in flow cytometry and other immunoassays to detect and quantify CCR2-positive cells.

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Lab products found in correlation

Anti mouse alexa fluor 568, by Thermo Fisher Scientific (2 mentions) Anti mouse alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Cd11b apc cy7, by BD (2 mentions) Anti rat alexa fluor 568, by Thermo Fisher Scientific (2 mentions) F4 80, by Thermo Fisher Scientific (2 mentions) Evos fl auto imaging system, by Thermo Fisher Scientific (2 mentions) Cd69 fitc, by BioLegend (2 mentions) Proliferating cell nuclear antigen (pcna), by BioLegend (2 mentions) Arginase 1, by Santa Cruz Biotechnology (2 mentions) Alexa fluor 488 anti rabbit, by Thermo Fisher Scientific (2 mentions) Pyvmt, by Abcam (2 mentions) F4 80, by Abcam (2 mentions) Cd40 apc, by BioLegend (1 mentions) Percp streptavidin, by BioLegend (1 mentions) Cd11b fitc, by BioLegend (1 mentions) Cd206 fitc, by BioLegend (1 mentions) Ly6c fitc, by BioLegend (1 mentions) Fix perm kit, by Thermo Fisher Scientific (1 mentions) B220 apc ra3 6b2, by BioLegend (1 mentions) Accuri c6 cytometer, by BD (1 mentions)

6 protocols using ccr2 fitc

1

Comprehensive Immune Cell Profiling by Flow Cytometry

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Mononuclear cells were stained with fluorescently labeled antibodies to CD11c-PE (HL3, BD Biosciences), CD4-FITC (RM4-4, Biolegend, San Diego, CA, USA), B220-biotin (RA3-6B2, Biolegend), B220-APC (RA3-6B2, Biolegend), I-A/I-E-APC (MHC class II, M5/114.15.2, Biolegend), CD40-APC (3/23, Biolegend), CD80-FITC (16-10A1, Biolegend), CD86-FITC (GL-1, Biolegend), CD3ε-biotin (145-2C11), Gr-1-biotin (RB6-8C5, Biolegend), TER119-biotin (TER-119, Biolegend), F4/80-FITC (BM8, Biolegend), CD11b-FITC (M1/70, Biolegend), CD107b-FITC (M3/84, Biolegend), FcεR1α-FITC (MAR-1, Biolegend), CD206-FITC (C068C2, Biolegend), Ly6c-FITC (HK1.4, Biolegend), CCR2-FITC (SA203G11, Biolegend), and CCR7-AlexaFluor488 (4B12, Biolegend). PerCP Streptavidin (Biolegend) was also used to stain the cells treated with biotinylated mAbs. For intracellular staining experiment, FIX & PERM Kit (Thermo Fisher Scientific) was used to stain CD4 T cells with mAb to FoxP3-PE (MF-14, Biolegend). After washing twice with buffer (PBS buffer containing 2% FBS, 2 mM EDTA, and 0.02% sodium azide), the expression level was analyzed by flow cytometry using a BD Accuri C6 cytometer (BD Biosciences). Data analysis was done by using BD Accuri C6 software.
Darkwah S., Nago N., Appiah M.G., Myint P.K., Kawamoto E., Shimaoka M, & Park E.J. (2019). Differential Roles of Dendritic Cells in Expanding CD4 T Cells in Sepsis. Biomedicines, 7(3), 52.
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2

Multimodal Immunophenotyping of 3D Cultures

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Cells were fixed in neutral formalin buffer and permeabilized with methanol. 3D cultures or carcinoma tissues were immunostained using procedures described4 . Samples were incubated with antibodies (1:100) to: CCR2-FITC (Biolegend cat no. 150607), F4/80 (Abcam, cat no. ab6640), CD8 (Biolegend cat no.100715), CD69-FITC (Biolegend cat no.104505), CD11b-APC-Cy7 (BD Pharmingen cat. no.557657), Arginase-1 (Santa Cruz Biotechnology cat no.sc20150), PyVmT (Abcam cat no. 73989) or PCNA (Biolegend cat no.307902) at 4°C overnight. CD8 and F4/80 were detected with anti-rat-Alexa Fluor-568 (Invitrogen cat no.A-11077). Arginase-I was detected with anti-rabbit-Alexa Fluor-488 (Invitrogen cat no.A-11034). PyVmT was detected with anti-mouse Alexa Fluor-568 (Invitrogen cat no. A-11004). PCNA was detected with anti-mouse-Alexa Fluor-488 (Invitrogen cat no. A-11001). Samples were counterstained with DAPI and mounted using PBS:glycerol. 5 images/field were captured at 10x magnification using the EVOS FL Auto Imaging System (Invitrogen). Expression was quantified by ImageJ as described75 (link).
Brummer G., Fang W., Smart C., Zinda B., Alissa N., Berkland C., Miller D, & Cheng N. (2019). CCR2 signaling in breast carcinoma cells promotes tumor growth and invasion by promoting CCL2 and suppressing CD154 effects on the angiogenic and immune microenvironments. Oncogene, 39(11), 2275-2289.
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3

Multimodal Immunophenotyping of 3D Cultures

Cited 1 time
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Cells were fixed in neutral formalin buffer and permeabilized with methanol. 3D cultures or carcinoma tissues were immunostained using procedures described4 . Samples were incubated with antibodies (1:100) to: CCR2-FITC (Biolegend cat no. 150607), F4/80 (Abcam, cat no. ab6640), CD8 (Biolegend cat no.100715), CD69-FITC (Biolegend cat no.104505), CD11b-APC-Cy7 (BD Pharmingen cat. no.557657), Arginase-1 (Santa Cruz Biotechnology cat no.sc20150), PyVmT (Abcam cat no. 73989) or PCNA (Biolegend cat no.307902) at 4°C overnight. CD8 and F4/80 were detected with anti-rat-Alexa Fluor-568 (Invitrogen cat no.A-11077). Arginase-I was detected with anti-rabbit-Alexa Fluor-488 (Invitrogen cat no.A-11034). PyVmT was detected with anti-mouse Alexa Fluor-568 (Invitrogen cat no. A-11004). PCNA was detected with anti-mouse-Alexa Fluor-488 (Invitrogen cat no. A-11001). Samples were counterstained with DAPI and mounted using PBS:glycerol. 5 images/field were captured at 10x magnification using the EVOS FL Auto Imaging System (Invitrogen). Expression was quantified by ImageJ as described75 (link).
Brummer G., Fang W., Smart C., Zinda B., Alissa N., Berkland C., Miller D, & Cheng N. (2019). CCR2 signaling in breast carcinoma cells promotes tumor growth and invasion by promoting CCL2 and suppressing CD154 effects on the angiogenic and immune microenvironments. Oncogene, 39(11), 2275-2289.
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4

Flow Cytometry Analysis of Immune Cell Subsets

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Flow cytometry was performed as described (6 (link)). Cells were incubated with anti-mouse CD16/32 (Fc Block; BD Biosciences) before staining with primary antibody or isotype controls. Ten-thousand to 50,000 events per sample were acquired using an LSRII flow cytometer (BD-Biosciences) and analyzed with Flowjo flow cytometry software (Tree Star Inc.). The following antibodies were used: CD11b-Brilliant Violet 421, Tim4-PE, CD138-APC, CD138-PE F4/80- Pacific Blue, Ly6G-APC-Cy7, Ly6C-APC-Cy7, CD80-PerCp-Cy5, CD40-PerCP-Cy5, CD206- PerCP-Cy5, CCR2-FITC, CX3CR1-FITC, CD169-APC, CCR5-APC, CD36-PE, CD36-AlexaFluor 488, and IL-10R-PE (Biolegend); Marco-FITC (Biorad); Ly6C-FITC, CD86-FITC, CD11c-FITC, I-A/I-E-PE, CD93-PE (BD Bioscience); CREB-PE and Phospho-CREB-FITC (Cell Signaling); CD11b-Pacific Blue, CD45-FITC, TLR4-PE, CD45-FITC (eBioscience). Buffers used in intracellular staining were from eBioscience. For cell-sorting, PEC from untreated B6 mice were stained with anti-CD11b, Tim4, and CD138 antibodies. CD11b+Tim4+ and CD11b+CD138+ cells were gated and sorted (FACSaria cell sorter). Forty-thousand cells/subset were collected and lysed immediately for RNA extraction.
Han S., Zhuang H., Shumyak S., Wu J., Li H., Yang L.J, & Reeves W.H. (2017). A novel subset of anti-inflammatory CD138+ macrophages is deficient in mice with experimental lupus*. Journal of immunology (Baltimore, Md. : 1950), 199(4), 1261-1274.
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5

Isolation and Characterization of Peritoneal Immune Cells

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Peritoneal exudate cells (PECs) were isolated via lavage and single cell suspensions were briefly washed with PBS prior to red blood cell lysis using ACK lysis buffer. Prepared cells were stained with Zombie Yellow (BioLegend) to assess live status of cells. The following antibodies were used, though not necessarily all in the same panel: F4/80- BV785 (BioLegend; Clone BM8), CD115-BV421 (BioLegend; Clone AFS98), CD11c-BV421 (BD Bioscience; Clone HL3), CD115-BUV395 (BD Bioscience; Clone AFS98), CD19-BUV737 (BD Bioscience; Clone 1D3), CD226-PE (BD Bioscience; Clone TX42.1), CD11b-PE (eBioscience; Clone M1/70), MHCII (I-A/I-E)- PE/Cy7 (BioLegend; Clone M5/114.15.2), Ly6G-PE/CF594 (BD Bioscience; Clone 1A8), Ly6C-APC/eFluor780 (eBiosceince; Clone HK1.4), CD102-Alexa Fluor 647 (BioLegend; Clone 3C4 (mlC2/4)), CD102-Alexa Fluor 488 (eBioscience; Clone 3C4 [mlC2/4]), CCR2-FITC (BioLegend; Clone SA203G11), and CD11b-PErCP/Cy5.5 (BioLegend; Clone M1/70). Antibody cocktails were prepared in FACs staining buffer (phosphate-buffered saline, 1% FBS, 1% 0.5mM EDTA). Cellular fluorescence was measured using an LSRII Fortessa flow cytometer, and data were analyzed using FlowJo software (Tree Star).
Martin A.T., Giri S., Safronova A., Eliseeva S.I., Kwok S.F, & Yarovinsky F. (2024). Parasite-induced IFN-γ regulates host defense via CD115 and mTOR-dependent mechanism of tissue-resident macrophage death. PLOS Pathogens, 20(2), e1011502.
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6

Phagocytosis Assay for Neutrophils

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Lithium-heparinized blood from BALB/c WT and Ncf1** mice or human NHD and SLE/CGD patients was mixed with SNECs in a 1:2 ratio and incubated at 37 °C. In some experiments, the blood was preincubated with varying concentrations of H2O2 (10 min), of the aminoferrocene compound MIS43 (10 min) [32 (link)], varying concentrations of catalase (Sigma) or butylated hydroxyanisole (BHA, Sigma) for 15 min before adding SNECs. Cells were stained with anti-mouse CD11b-eFluor450 (eBioscience), anti-mouse Ly6C-APC (BioLegend), Ly6G-PE/Cy7 (BioLegend), CCR2-FITC (BioLegend), I-A/I-E (MHC II)-Alexa Fluor 700 (BioLegend) or anti-human CD14-eFluor450 (eBioscience), CD16-APC/Cy7 and CCR2-PerCP/Cy5.5 (BioLegend), respectively. Samples were subjected to hypotonic water lysis of erythrocytes and analyzed using a Beckman Coulter Gallios™ flow cytometer. The Phagocytosis index (PhIx) was calculated in the following way: PhIx = (%PI/pHrodo-positive phagocytes x MFIPI/pHrodo). For the analysis of cytokine/chemokine production upon phagocytosis, plasma was isolated from blood incubated with SNECs (3 h) and subjected to LegendPlex bead-based assay (BioLegend).
Hahn J., Euler M., Kilgus E., Kienhöfer D., Stoof J., Knopf J., Hahn M., Harrer T., Hultqvist M., Olofsson P., Mokhir A., Holmdahl R., Herrmann M., Schett G., Muñoz L.E, & Hoffmann M.H. (2019). NOX2 mediates quiescent handling of dead cell remnants in phagocytes. Redox Biology, 26, 101279.
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