Brain single-cell suspensions from 9-mo-old WT mice were stained with Ghost Dye violet 510 (1:1,000; Tonbo Biosciences) and anti–CD16/CD32 antibody (1:100; BD Biosciences) followed by anti-CD11b (1:100; BioLegend), anti-CD45 (1:100; BioLegend), anti-CD11c (1:50; BioLegend), anti-TMEM119 (1:200; Abcam), and anti-CCR2 (1:100; BioLegend). CD11b+ cells were gated from single/live cells followed by subsequent gating of CD11b+CD45low as microglia and CD11b+CD45high as macrophages. To distinguish microglia and macrophages, the CCR2 marker expressed by blood-derived macrophages but not by microglia was used (3 (link), 16 (link)). To further distinguish these two cell types, the microglia-specific marker Tmem119 (17 (link)) was also included. The CD11b+CD45high cells that express CCR2 but not Tmem119 were confirmed as macrophages, while the CD11b+CD45low cells that all express Tmem119 but do not express CCR2 were confirmed as microglia. FMO negative controls were included to confirm the specificity of CD11c staining in CD11b+CD45low microglial populations. Brain CD45− cells that mainly contain nonimmune cells (e.g., neurons, astrocytes, and oligodendrocytes) that do not express CD11c were also included as negative controls to further validate the specificity of this strategy.
Anti ccr2
Manufactured by BioLegend
Anti-CCR2 is a laboratory reagent that targets the chemokine receptor CCR2. CCR2 is a G-protein coupled receptor that plays a role in the migration and activation of monocytes and other immune cells. Anti-CCR2 can be used to detect and study the expression of CCR2 in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd45, by BioLegend (3 mentions)
Anti cd11b, by BioLegend (3 mentions)
Anti cd11c, by BioLegend (2 mentions)
Ghost dye violet 510, by Cytek Biosciences (1 mentions)
Anti tmem119, by Abcam (1 mentions)
Anti cd16 cd32 antibody, by BD (1 mentions)
Anti f4 80, by BioLegend (1 mentions)
Anti cd206, by BioLegend (1 mentions)
Anti ly6g ly6c, by BioLegend (1 mentions)
Anti cd115, by BioLegend (1 mentions)
Facsaria 2, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd16 32 fc block, by BD (1 mentions)
Anti sirpα, by BioLegend (1 mentions)
Sh800 cell sorter, by Sony (1 mentions)
Transwell insert, by Corning (1 mentions)
Anti ly6g, by BioLegend (1 mentions)
Facs lysis solution, by BD (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Anti cd11b 647, by Thermo Fisher Scientific (1 mentions)
5 protocols using anti ccr2
1
Distinguishing Microglia and Macrophages
2
Multi-marker Profiling of Immune Cells
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Isolated SVF cells or leukocytes were resuspended in 200 µl PBS containing 0.25% BSA, 0.2 mM EDTA, and 1% penicillin/streptomycin. Cells were preincubated for 7 min at 4 °C in Fc Block (CD16/32, BD Biosciences) and then stained for 15 min with fluorophore-conjugated antibodies at 4 °C. The following antibodies were used: anti-CD45 (clone: 30-F11, Biolegend), anti-F4/80 (clone: BM8, Biolegend), anti-CD11b (clone: M1/70, Biolegend), anti-CD11c (clone: N418, Biolegend), anti-CD206 (clone: C068C2, Biolegend), anti-CCR2 (clone: SA203G11, Biolegend), anti-Ly6G/Ly6C (clone: RB6-8C5, Biolegend), and anti-CD115 (clone: AFS98, Biolegend)15 (link). Flow cytometric analysis was performed using a FACSCantoII (BD Biosciences). Cell sorting was performed with a FACSAriaII (BD Biosciences). Data were analyzed using FlowJo software (v9.4.10, Tree Star).
3
Isolation and Co-culture of Bone Marrow Macrophages
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BM cells isolated from WT C57BL/6 mice and Tram–/– mice (CD45.2+) were cultured with complete RPMI 1640 medium supplemented with M-CSF (10 ng/mL) for 3 days. The cells were harvested, stained with anti-Ly6G (1:200 dilution, BioLegend, no. 127606) and anti-CD11b (1:200 dilution, BioLegend, no. 101226) antibodies, and labeled with PI. CD11b+Ly6G–PI– cells were purified with SH800 cell sorter (Sony) to obtain donor BMMs. BM cells isolated from B6 SJL mice (CD45.1+) were cultured in 12-well plates (5 × 105 cells/well) with complete RPMI 1640 medium supplemented with M-CSF (10 ng/mL) to serve as recipient cells. After 3 days, floating cells were removed, and Transwell inserts (Corning) were placed in some recipient cell cultures. WT or Tram–/– donor BMMs were added to the upper chambers of the Transwell inserts (5 × 105 cells/well) or directly added to the recipient BMMs without Transwell inserts (5 × 105 cells/well). After 2 days, the cells were harvested and stained with anti-CD45.1 (1:200 dilution, BioLegend, no. 110730), anti-CCR2 (1:200 dilution, BioLegend, no. 150628), anti–SIRP-α (1:200 dilution, BioLegend, no. 144014), and anti-CD200R (1:200 dilution, BioLegend, no. 123916) antibodies. The surface phenotype of CD45.1+ recipient BMMs was determined with flow cytometry.
4
Characterizing Murine Myeloid Cell Populations
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Blood and BM samples from killed mice were blocked for 15 min at 4 °C in PBS containing 5% BSA and a 1:100 dilution of anti-CD16/CD32 (24g2, BD Pharmingen, 553142). Samples were then stained for 30 min at 4 °C with anti-CD11b 647 (eBiosciences, 51-0112-82) or biotinylated (BD Pharmingen, 51.01712 J), anti-CD45 V450 (eBiosciences, 48-0451-82), anti-Ly6C FITC (BD Bioscience, 553104) or APC (BD Pharmingen, 560595), anti-CCR5 PE (eBioscience, 12.1951-12), and anti-CCR2 (Biolegend, 150607) primary antibodies and with streptavidin PE (BD Bioscience, 554061) secondary reagent. Erythrocytes in blood samples were lysed with FACS Lysis Solution (BD Biosciences, 349202) for 7 min at room temperature. Before gating, granulocytes were excluded by FCS/SSC. Peritoneal macrophages were collected on the indicated days after TG administration and blocked with BSA/anti-CD16/CD32. Macrophages were then stained for 30 min at 4 °C with anti-CD11b 647 (eBiosciences, 51-0112-82), anti-CD45 V450 (eBiosciences, 48-0451-82), anti-F4/80 Pe-Cy7 (Biolegend, 123114), anti-AIM (GeneTex, GTX37448), and anti-CD36 (Cascade BioScience, ABM-5525) antibodies; for quantification of dead cells, Hoechst 33258 (Sigma, 861405) was added 5 min previous to flow cytometry analysis. Data were acquired in a FACSCanto III cytometer (BD) and analyzed using FlowJo software (Tree Star).
5
Protein Interaction Analysis via Duolink PLA
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Protein interactions were assessed using the Duolink PLA assay (Sigma). Briefly, cells were fixed with 10% neutral buffer formalin, permeabilized with PBS/0.2% Triton X-100 and incubated with the following antibody pairs: anti-CCR2 (1:100 dilution, Biolegend, cat no.357202) and anti-SRC (1:100, Cell Signaling Technology, cat no.2123S), anti-MET (1:50, Santa Cruz Biotechnology, cat no.SC-514148) and anti-SRC, or anti-CCR2 and anti-MET (1:100, Cell Signaling Technology, cat no.4560). Samples were incubated with Duolink in situ Probes; anti-rabbit Minus (cat no.DUO92005) and anti-mouse PLUS (cat no.DUO92001). Signals were amplified with polymerase using In Situ Detection Reagents Green (cat no.DUO92014). Duolink PLA wash buffers (cat no.DUO82049) were used. Cells were counterstained with DAPI. Images were captured at 10x magnification using the FL-Auto EVOS imager.
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