Anti ccr2 pe

Manufactured by R&D Systems

Sourced in United States

Anti-CCR2-PE is a laboratory reagent used for the detection and quantification of the CCR2 receptor by flow cytometry. CCR2 is a chemokine receptor that plays a role in the migration and activation of monocytes, macrophages, and other immune cells. This reagent consists of an anti-CCR2 antibody conjugated to the fluorescent dye phycoerythrin (PE).

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3 protocols using anti ccr2 pe

Antibody Staining and Cell Cycle Analysis

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For antibody staining, 200,000 cells were seeded in 6-cm dishes overnight. Cells were detached from plates with Accutase (EMD Millipore cat #SCR005) at 37^oC for 5 minutes, washed in PBS and incubated with anti-CCR2-PE diluted 1:50, (R&D Systems, cat# FAB151P) for 1 hour. For cell cycle studies, 200,000 cells were seeded in 6-cm dishes overnight. Cells were treated with or without 2mM Thymidine (Sigma, cat # T1895–1G) for 16 hours. The cells were washed 3 times with serum free media (BT-20: EMEM, HCC1937: RPMI, and MCF10CA1d: DMEM). Growth media was added for 2 hours for MCF10CA1d cells and 10 hours for BT-20 and HCC1937 cells. Cells were incubated with 2mM Thymidine for 16 hours, washed with serum free media and incubated with growth media, with or without 100 ng/ml CCL2. Cells were detached with Accutase and fixed with 70% ethanol at −20ᵒC. Samples were incubated in 500 μL of PBS/0.1% Triton X-100/2mg/ml RNase A (VWR, cat# 97064–064) and 200 μg/ml propidium iodide (Invitrogen, cat# P3566) for 15 minutes at 37ᵒC.
For ALDH activity assay, cells were seeded in 6 well plates (200,000/well), serum starved for 24 hours and incubated in serum free medium with or without 100 ng/ml CCL2 for 24 hours. Cells were subject to AldeRed™ Assay (EMT Millipore, cat #SCR150) according to commercial protocol. All cells were analyzed using a BD LSRII Flow Cytometer.

Yao M., Fang W., Smart C., Hu Q., Huang S., Alvarez N., Fields P, & Cheng N. (2018). CCR2 chemokine receptors enhance growth and cell cycle progression of breast cancer cells through SRC and PKC activation. Molecular cancer research : MCR, 17(2), 604-617.

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Flow Cytometry Analysis of Immune Cells

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Cell suspensions (1×10⁷ cells in 100 µl) were incubated with Fc Block (cat. no. 101319; 1:100; BioLegend, Inc., San Diego, CA, USA) at 4°C for 5 min and labeled with the following fluorescently conjugated antibodies: Anti-CD45 APC (cat. no. 103111; 1:100) anti-Ly-6 G PerCP/Cy5.5 (cat. no. 127165; 1:100); anti-F4/80 PE/Cy7 (cat. no. 123113; 1:100); anti-MHC-II FITC (cat. no. 116405; 1:100) all obtained from BioLegend, Inc. and anti-CCR2 PE (cat. no. FAB5538P; R&D systems, Minneapolis, MN, USA) for 30 min at 4°C. Cells were washed twice in FACS buffer. Flow cytometry analysis was performed on a flow cytometer (BD FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) and data analysis was performed using the FlowJo 10.0 software (Tree Star, Inc., Ashland, OR, USA).

Liu B., Zhang H.G., Zhu Y., Jiang Y.H., Luo G.P., Tang F.Q., Jian Z, & Xiao Y.B. (2017). Cardiac resident macrophages are involved in hypoxia-induced postnatal cardiomyocyte proliferation. Molecular Medicine Reports, 15(6), 3541-3548.

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Murine Liver Cell Isolation and Neutrophil Identification

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Single cell suspensions were generated from murine livers as previously described (16) .
Suspensions at a concentration of 10 7 cells/ml were incubated at room temperature (45 mins) in combinations of the following antibodies in the presence of Fc blocking antibody: anti-CD45-PE, anti-Ly6G(Gr1)-PE, anti-CD11b-PECy7, anti-CCR5-PE, anti-CXCR3-APC, anti-CXCR4-APC, anti-CXCR5-APC, anti-CD19-PE, anti-CD3-APC, anti-NK1.1-APC (all eBioscience), anti-CCR1-PE, anti-CCR2-PE, anti-CXCR2-APC, anti-CXCR6-APC, anti-CXCR7-APC (all R&D systems), anti-CCR3-PE and anti-CCR4-PE (both Biolegend). Analysis was performed using FlowJo software. The percentage of positive cells within samples was determined and the total number of cells of interest per organ was calculated.
For sorting of CD45 + /CXCR2 + neutrophils from normal and tumor-bearing livers, single cell hepatic suspensions pooled from 6 tumor-bearing or control mice were stained with anti-CD45-PE and anti-CXCR2-APC as above, before being FACS sorted using a high speed cell sorter (Beckman Coulter).
In-vivo administration of the neutrophil depleting antibody 1A8 may mask the Ly6G epitope making it impossible to subsequently identify neutrophils. We therefore used a combination of CD45 and CXCR2 to identify neutrophils in animal experiments where neutrophil depletion was performed.

Gordon-Weeks A.N., Lim S.Y., Yuzhalin A.E., Jones K., Markelc B., Kim K.J., Buzzelli J.N., Fokas E., Cao Y., Smart S, & Muschel R. (2017). Neutrophils promote hepatic metastasis growth through fibroblast growth factor 2-dependent angiogenesis in mice. Hepatology (Baltimore, Md.), 65(6).

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