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mouse antibodies against human chemokines ccl2 (R&D Systems)

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R&D Systems mouse antibodies against human chemokines ccl2

a Culture medium collected from the upper and lower wells of transwells with HUVEC monolayers either not stimulated (NS) or stimulated with TNF-α + IFN-γ were assayed for <t>CCL2,</t> CCL7, and CCL8 by ELISA ( n = 3 separate experiments). Each symbol shows data from one experiment, with assays done in duplicate, and bars indicate means ± SEM. b Confocal microscopy images at × 40 magnification of permeabilized and non-permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL2, CCL7, CCL8, <t>CCL5,</t> CCL20, and CXCL9 (green) and stained using phalloidin for polymerized actin (magenta) and DAPI for nuclei (blue). The scale bars indicate 10 µm. Images are representative of three experiments. c Biotinylated CCL5 (0.1 μM) and CCL2 (1 μM) were incubated with CHO-K1 cells and the heparan-sulfate deficient cell line, D-677. Chemokine binding was detected using streptavidin-phycoerythrin (PE) and flow cytometry. Data shown are representative of three separate experiments.

Mouse Antibodies Against Human Chemokines Ccl2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 59 article reviews

mouse antibodies against human chemokines ccl2 - by Bioz Stars, 2026-09

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Images

1) Product Images from "Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines"

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

Journal: Nature Communications

doi: 10.1038/s41467-025-57002-6

Figure Legend Snippet: a Culture medium collected from the upper and lower wells of transwells with HUVEC monolayers either not stimulated (NS) or stimulated with TNF-α + IFN-γ were assayed for CCL2, CCL7, and CCL8 by ELISA ( n = 3 separate experiments). Each symbol shows data from one experiment, with assays done in duplicate, and bars indicate means ± SEM. b Confocal microscopy images at × 40 magnification of permeabilized and non-permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL2, CCL7, CCL8, CCL5, CCL20, and CXCL9 (green) and stained using phalloidin for polymerized actin (magenta) and DAPI for nuclei (blue). The scale bars indicate 10 µm. Images are representative of three experiments. c Biotinylated CCL5 (0.1 μM) and CCL2 (1 μM) were incubated with CHO-K1 cells and the heparan-sulfate deficient cell line, D-677. Chemokine binding was detected using streptavidin-phycoerythrin (PE) and flow cytometry. Data shown are representative of three separate experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Incubation, Binding Assay, Flow Cytometry

For ( a , c ), human CD4 + T cell subgroups were isolated by FACS from the blood of healthy donors as in Supplementary Fig. and Fig. . a CCR6 +(high) CCR2 + cells were either left untreated (marked with a minus sign) or treated with CCL2 (100 ng/ml) just before and for four min after adding the cells to the flow chambers, and CCL2-treated cells were used without (marked with a minus sign) or with treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested, and transmigrated, while the middle panel shows arrested cells as percentages of cells rolling and the right panel shows transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with n = 4 individual donors in four separate experiments using CCL2. Two of these experiments included the additional treatment with BMS22. b Above is the CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black, and C-terminal GAG-binding sequence of CXCL9 in red. Below are confocal microscopy images of control or CCL2-CXCL9-chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and stained with DAPI for nuclei (blue). Images are representative of three experiments. c Numbers of CCR6 +(high) CCR2 + cells, either untreated (marked with a minus sign) or treated with the CCR2 antagonist, BMS22, rolling, arrested, and transmigrated on TNF- α -activated HUVECs transduced with either control virus (marked with a minus sign) or virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as percentages of cells rolling and in the right panel transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with cells from four donors and separate experiments in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means ± SEM. p values were calculated using two-tailed paired Student’s t tests, and no corrections were made for multiple comparisons. Source data are provided in the file, and representative videos used to quantify rolling, arrest, and transendothelial migration are provided in Supplementary Movies – .

Figure Legend Snippet: For ( a , c ), human CD4 + T cell subgroups were isolated by FACS from the blood of healthy donors as in Supplementary Fig. and Fig. . a CCR6 +(high) CCR2 + cells were either left untreated (marked with a minus sign) or treated with CCL2 (100 ng/ml) just before and for four min after adding the cells to the flow chambers, and CCL2-treated cells were used without (marked with a minus sign) or with treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested, and transmigrated, while the middle panel shows arrested cells as percentages of cells rolling and the right panel shows transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with n = 4 individual donors in four separate experiments using CCL2. Two of these experiments included the additional treatment with BMS22. b Above is the CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black, and C-terminal GAG-binding sequence of CXCL9 in red. Below are confocal microscopy images of control or CCL2-CXCL9-chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and stained with DAPI for nuclei (blue). Images are representative of three experiments. c Numbers of CCR6 +(high) CCR2 + cells, either untreated (marked with a minus sign) or treated with the CCR2 antagonist, BMS22, rolling, arrested, and transmigrated on TNF- α -activated HUVECs transduced with either control virus (marked with a minus sign) or virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as percentages of cells rolling and in the right panel transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with cells from four donors and separate experiments in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means ± SEM. p values were calculated using two-tailed paired Student’s t tests, and no corrections were made for multiple comparisons. Source data are provided in the file, and representative videos used to quantify rolling, arrest, and transendothelial migration are provided in Supplementary Movies – .

Techniques Used: Isolation, Sequencing, Binding Assay, Confocal Microscopy, Control, Staining, Transduction, Virus, Two Tailed Test, Migration

Cytokine-activated endothelial cells upregulate expression of selectins and integrin ligands and secrete chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic-like type 17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5, and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are removed by blood flow to create a transendothelial gradient that drives TEM. Although for the sake of clarity the T cell depicted here co-expresses the four chemokine receptors, not all CCR6 +(high) CCR2 + CD4 + T cells express CCR5 and/or CXCR3.

Figure Legend Snippet: Cytokine-activated endothelial cells upregulate expression of selectins and integrin ligands and secrete chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic-like type 17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5, and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are removed by blood flow to create a transendothelial gradient that drives TEM. Although for the sake of clarity the T cell depicted here co-expresses the four chemokine receptors, not all CCR6 +(high) CCR2 + CD4 + T cells express CCR5 and/or CXCR3.

Techniques Used: Expressing, Binding Assay

Migration:

Article Title: Wnt signaling regulates chemokine production and cell migration of circulating human monocytes
Article Snippet: .. In some experiments, the media were pre-incubated with the neutralizing antibody against human CCL2 (R&D Systems MAB679, 2 μg/ml) for 1.5 h at room temperature prior to being used in the lower chambers of the migration assays. .. All statistical analyses used the recommended tests and GraphPad Prism 9.

Article Title: Soft extracellular matrix enhances inflammatory activation of mesenchymal stromal cells to induce monocyte production and trafficking
Article Snippet: .. In some experiments, the conditioned media were incubated with the neutralizing antibody against human CCL2 (MAB679, R&D Systems) or the mouse IgG2B isotype control (MAB004, R&D Systems) for 3 hours at 4°C prior to the migration assay. ..

Staining:

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity
Article Snippet: .. Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona). .. The tissue array was scanned and images viewed using the Olympus OlyVIA software 2.3 (Build 8529, Olympus America Inc.).

Incubation:

Article Title: Systemic Inflammation in Metabolic Syndrome: Increased Platelet and Leukocyte Activation, and Key Role of CX 3 CL1/CX 3 CR1 and CCL2/CCR2 Axes in Arterial Platelet-Proinflammatory Monocyte Adhesion
Article Snippet: Leukocyte-Endothelial Cell interactions Under Flow Conditions Before starting each assay, whole blood was diluted 1:10 with Hank’s balanced salt solution (HBSS; Lonza, Barcelona, Spain) without calcium or magnesium at 37oC. .. Blood was perfused across HUAEC monolayers, unstimulated or stimulated with 20 ng/mL TNFα (SigmaAldrich, Madrid, Spain) for 24 h. Some cells were incubated with a monoclonal neutralizing antibody against human CX3CL1 (5 μg/mL, IgG1; ref: MAB3652, R&D Systems, Abingdon, UK), neutralizing antibody against human CCL2 (2 μg/mL, IgG1; ref: MAB279, R&D Systems) or with an isotype-matched control antibody (MOPC-21, 2 μg/mL, IgG1; ref: M5284, Sigma-Aldrich) 10 min before blood perfusion. ..

Control:

Image Search Results

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: a Culture medium collected from the upper and lower wells of transwells with HUVEC monolayers either not stimulated (NS) or stimulated with TNF-α + IFN-γ were assayed for CCL2, CCL7, and CCL8 by ELISA ( n = 3 separate experiments). Each symbol shows data from one experiment, with assays done in duplicate, and bars indicate means ± SEM. b Confocal microscopy images at × 40 magnification of permeabilized and non-permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL2, CCL7, CCL8, CCL5, CCL20, and CXCL9 (green) and stained using phalloidin for polymerized actin (magenta) and DAPI for nuclei (blue). The scale bars indicate 10 µm. Images are representative of three experiments. c Biotinylated CCL5 (0.1 μM) and CCL2 (1 μM) were incubated with CHO-K1 cells and the heparan-sulfate deficient cell line, D-677. Chemokine binding was detected using streptavidin-phycoerythrin (PE) and flow cytometry. Data shown are representative of three separate experiments.

Article Snippet: Samples were incubated at room temperature for 2 h with primary mouse antibodies against human chemokines CCL2 (5 μg, Cat# MAB679) CCL5 (5 μg, Cat# MAB278), CCL7 (5 μg, Cat# MAB282), CCL8 (5 μg, Cat# MAB281), CCL20 (5 μg, Cat# AF360), CXCL9 (5 μg, Cat# MAB392) from R&D Systems or with mouse IgG 1 isotype control (5 μg, Cat# MAB002) or mouse IgG 2B isotype control (Cat# MAB004) from R&D Systems, or with anti-human GOLPH2 (2 μg, Cat# PA5-30622) or rabbit IgG isotype control from Invitrogen, or with anti-human vWF (2 μg, Cat# ab6994) from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Incubation, Binding Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: For ( a , c ), human CD4 + T cell subgroups were isolated by FACS from the blood of healthy donors as in Supplementary Fig. and Fig. . a CCR6 +(high) CCR2 + cells were either left untreated (marked with a minus sign) or treated with CCL2 (100 ng/ml) just before and for four min after adding the cells to the flow chambers, and CCL2-treated cells were used without (marked with a minus sign) or with treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested, and transmigrated, while the middle panel shows arrested cells as percentages of cells rolling and the right panel shows transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with n = 4 individual donors in four separate experiments using CCL2. Two of these experiments included the additional treatment with BMS22. b Above is the CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black, and C-terminal GAG-binding sequence of CXCL9 in red. Below are confocal microscopy images of control or CCL2-CXCL9-chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and stained with DAPI for nuclei (blue). Images are representative of three experiments. c Numbers of CCR6 +(high) CCR2 + cells, either untreated (marked with a minus sign) or treated with the CCR2 antagonist, BMS22, rolling, arrested, and transmigrated on TNF- α -activated HUVECs transduced with either control virus (marked with a minus sign) or virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as percentages of cells rolling and in the right panel transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with cells from four donors and separate experiments in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means ± SEM. p values were calculated using two-tailed paired Student’s t tests, and no corrections were made for multiple comparisons. Source data are provided in the file, and representative videos used to quantify rolling, arrest, and transendothelial migration are provided in Supplementary Movies – .

Techniques: Isolation, Sequencing, Binding Assay, Confocal Microscopy, Control, Staining, Transduction, Virus, Two Tailed Test, Migration

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: Cytokine-activated endothelial cells upregulate expression of selectins and integrin ligands and secrete chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic-like type 17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5, and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are removed by blood flow to create a transendothelial gradient that drives TEM. Although for the sake of clarity the T cell depicted here co-expresses the four chemokine receptors, not all CCR6 +(high) CCR2 + CD4 + T cells express CCR5 and/or CXCR3.

Techniques: Expressing, Binding Assay

Figure 1. Exposure to high altitude results in PH and increased secretion of inflammatory classical monocyte ligands from the lungs. (A) Schematic showing hypoxia exposure time course in wildtype mice. Duration of hypoxia exposure is directly proportional to (B) RVSP and RV hypertrophy as measured by Fulton Index (N=6-13/group). At 3 days of hypoxia, increased protein expression of classical monocyte ligands (C) CCL2 (N=6-11/group) and (D) CCL12 (N=6- 11/group), whereas significantly lower levels of nonclassical monocyte ligand (E) CX3CL1 (N=6/group) in the lungs. (F) Higher CCL2 gradient in lungs and in the (G) peripheral blood of wildtype mice following 3 days of hypoxia exposure (N=5/group). Data in all panels were obtained from female mice. Statistical analysis was conducted using ANOVA, followed by Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. N=number of animals, mean±SD, CI=confidence interval.

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 1. Exposure to high altitude results in PH and increased secretion of inflammatory classical monocyte ligands from the lungs. (A) Schematic showing hypoxia exposure time course in wildtype mice. Duration of hypoxia exposure is directly proportional to (B) RVSP and RV hypertrophy as measured by Fulton Index (N=6-13/group). At 3 days of hypoxia, increased protein expression of classical monocyte ligands (C) CCL2 (N=6-11/group) and (D) CCL12 (N=6- 11/group), whereas significantly lower levels of nonclassical monocyte ligand (E) CX3CL1 (N=6/group) in the lungs. (F) Higher CCL2 gradient in lungs and in the (G) peripheral blood of wildtype mice following 3 days of hypoxia exposure (N=5/group). Data in all panels were obtained from female mice. Statistical analysis was conducted using ANOVA, followed by Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. N=number of animals, mean±SD, CI=confidence interval.

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Expressing

Figure 4: Genetic and pharmacologic blockade of CCR2-CCL2 axis protects from hypoxic PH. (A) Schematic showing the BM reconstitution of Ccr2-/- and WT BM into lethally irradiated wildtype mice. Wildtype mice reconstituted with Ccr2-/- BM were protected from hypoxic PH by attenuated (B) RVSP (N=7-11/group) and (C) RV hypertrophy (N=7-11/group) as measured by Fulton Index, compared to wildtype mice that were reconstituted with wildtype BM. (D) Schematic showing pharmacological blockade of CCR2 ligands CCL2 or CCL7 using anti-CCL2 or anti-CCL7 neutralizing antibody treatment. Hypoxia exposed wildtype mice treated with CCL2 NAb but not CCL7 NAb showed lower (E) RVSP (N=6/group) and (F) RV hypertrophy (N=6/group). TSP-1 levels in (G) lungs (N=6/group) and (H) blood (N=6/group); and TGF-β1 levels in (I) lungs (N=6/group) and (J) blood (N=6/group) compared to wildtype mice treated with isotype control antibody. Data in all panels followed a normal distribution. ANOVA with the Tukey test was performed for multiple comparisons. Data were obtained from the female mice. mean ± SD

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 4: Genetic and pharmacologic blockade of CCR2-CCL2 axis protects from hypoxic PH. (A) Schematic showing the BM reconstitution of Ccr2-/- and WT BM into lethally irradiated wildtype mice. Wildtype mice reconstituted with Ccr2-/- BM were protected from hypoxic PH by attenuated (B) RVSP (N=7-11/group) and (C) RV hypertrophy (N=7-11/group) as measured by Fulton Index, compared to wildtype mice that were reconstituted with wildtype BM. (D) Schematic showing pharmacological blockade of CCR2 ligands CCL2 or CCL7 using anti-CCL2 or anti-CCL7 neutralizing antibody treatment. Hypoxia exposed wildtype mice treated with CCL2 NAb but not CCL7 NAb showed lower (E) RVSP (N=6/group) and (F) RV hypertrophy (N=6/group). TSP-1 levels in (G) lungs (N=6/group) and (H) blood (N=6/group); and TGF-β1 levels in (I) lungs (N=6/group) and (J) blood (N=6/group) compared to wildtype mice treated with isotype control antibody. Data in all panels followed a normal distribution. ANOVA with the Tukey test was performed for multiple comparisons. Data were obtained from the female mice. mean ± SD

Techniques: Irradiation, Control

Figure 5: Resident IMs are a major source of CCL2 and recruited IMs are a major source of pathologic TSP-1 in hypoxic PH. (A) Flow cytometry analysis using Ccl2RFP-flox reporter mice showed a higher number of CCL2+ IMs (N=14/group; N=14/group, 9F and 5M in Nx; 8F and 6M in Hx), and (B) FOLR2+ IMs are a major source of CCL2 (N=14/group). (C) Hypoxia exposed wildtype mice following intracellular CCL2 staining by flow cytometry also showed a higher number of CCL2+ IMs (N=7/group, female mice). (D). IM subpopulation analysis using flow

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 5: Resident IMs are a major source of CCL2 and recruited IMs are a major source of pathologic TSP-1 in hypoxic PH. (A) Flow cytometry analysis using Ccl2RFP-flox reporter mice showed a higher number of CCL2+ IMs (N=14/group; N=14/group, 9F and 5M in Nx; 8F and 6M in Hx), and (B) FOLR2+ IMs are a major source of CCL2 (N=14/group). (C) Hypoxia exposed wildtype mice following intracellular CCL2 staining by flow cytometry also showed a higher number of CCL2+ IMs (N=7/group, female mice). (D). IM subpopulation analysis using flow

Techniques: Flow Cytometry, Staining

Figure 8: DEX prophylaxis blunts CCL2 production by resident IMs and blocks the recruitment of TSP-1 producing CCR2+ IMs in hypoxia. (A) DEX prophylactically-treated, hypoxia-exposed Ccl2RFP-flox reporter mice exhibited a significant reduction in CCL2+ IMs, particularly in (B) CCL2RFP+ resident IMs (N=7/group). Additionally, (C) intracellular CCL2 flow cytometry analysis in DEX prophylactically-treated hypoxia-exposed wildtype mice revealed a

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 8: DEX prophylaxis blunts CCL2 production by resident IMs and blocks the recruitment of TSP-1 producing CCR2+ IMs in hypoxia. (A) DEX prophylactically-treated, hypoxia-exposed Ccl2RFP-flox reporter mice exhibited a significant reduction in CCL2+ IMs, particularly in (B) CCL2RFP+ resident IMs (N=7/group). Additionally, (C) intracellular CCL2 flow cytometry analysis in DEX prophylactically-treated hypoxia-exposed wildtype mice revealed a

Techniques: Flow Cytometry

Treating monocytes with Wnt-3a induces cell migration and CCL2 production. Naïve monocytes were plated in the top chamber of a transwell and exposed to different conditioned media (CM). Cells that crossed the transwell were collected from the bottom well and counted by flow cytometry. A Schematic illustration of experimental design. Naïve monocytes were exposed to Wnt-3a/control CM obtained from L-cells for 2 h (I). Naïve monocytes were exposed to Wnt-3a/control CM obtained from monocytes treated with Wnt-3a/control for 8 h (II). Treated monocytes (as in II) were washed and transferred to fresh RPMI medium for an additional 1 h. CM was collected and used to treat naïve monocytes in the transwell assay (III). B Flow cytometry counts of cells crossing the transwell. * P -value = 0.0307, *** P -value = 0.0003 and ** P -value = 0.0045 for paired t-tests. Y-axis represents the absolute cell count. C ELISA assays of CCL2 concentration in media used for the indicated experiments. Protein concentrations were calculated by regression from a standard curve. D Flow cytometry counts of cells crossing the transwell. Y-axis represents the absolute cell count. X-axis represents the media used as in I and II of the experimental design, with or without 2 μg/ml CCL2 inhibitor, marked as circles and squares, respectively. The white and black shapes represent each donor. FC – average fold change of the marked pairs. E A representative blot of media collected from the top and bottom chambers of the different experiments (as indicated), at the end of the transwell incubation period. Media were centrifuged to remove cells and analyzed by western blot using a specific anti-Wnt-3a antibody. C – control/control-treated media, W3 – Wnt-3a/Wnt-3a-treated media

Journal: Cell Communication and Signaling : CCS

Article Title: Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

doi: 10.1186/s12964-024-01608-8

Figure Lengend Snippet: Treating monocytes with Wnt-3a induces cell migration and CCL2 production. Naïve monocytes were plated in the top chamber of a transwell and exposed to different conditioned media (CM). Cells that crossed the transwell were collected from the bottom well and counted by flow cytometry. A Schematic illustration of experimental design. Naïve monocytes were exposed to Wnt-3a/control CM obtained from L-cells for 2 h (I). Naïve monocytes were exposed to Wnt-3a/control CM obtained from monocytes treated with Wnt-3a/control for 8 h (II). Treated monocytes (as in II) were washed and transferred to fresh RPMI medium for an additional 1 h. CM was collected and used to treat naïve monocytes in the transwell assay (III). B Flow cytometry counts of cells crossing the transwell. * P -value = 0.0307, *** P -value = 0.0003 and ** P -value = 0.0045 for paired t-tests. Y-axis represents the absolute cell count. C ELISA assays of CCL2 concentration in media used for the indicated experiments. Protein concentrations were calculated by regression from a standard curve. D Flow cytometry counts of cells crossing the transwell. Y-axis represents the absolute cell count. X-axis represents the media used as in I and II of the experimental design, with or without 2 μg/ml CCL2 inhibitor, marked as circles and squares, respectively. The white and black shapes represent each donor. FC – average fold change of the marked pairs. E A representative blot of media collected from the top and bottom chambers of the different experiments (as indicated), at the end of the transwell incubation period. Media were centrifuged to remove cells and analyzed by western blot using a specific anti-Wnt-3a antibody. C – control/control-treated media, W3 – Wnt-3a/Wnt-3a-treated media

Article Snippet: In some experiments, the media were pre-incubated with the neutralizing antibody against human CCL2 (R&D Systems MAB679, 2 μg/ml) for 1.5 h at room temperature prior to being used in the lower chambers of the migration assays.

Techniques: Migration, Flow Cytometry, Control, Transwell Assay, Cell Counting, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Wnt signaling regulates chemokine production and cell migration of circulating human monocytes

doi: 10.1186/s12964-024-01608-8

Figure Lengend Snippet:

Techniques: Sequencing, Amplification

(A) Culture supernatants collected from the upper and lower wells of transwells with HUVEC monolayers were assayed for CCL2, CCL7 and CCL8 by ELISA. Data are from three experiments and bars indicate means +/− SEM. (B) Confocal microscopy images at ×40 magnification of non-permeabilized and permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL20, CCL5, CXCL9, CCL2, CCL7 or CCL8 (green) along with nuclear staining (blue) as described in Material and methods. The scale bars indicate 10 µm. Images are representative of three experiments. (C) CCR6 + CCR2 + cells were either left untreated or treated with CCL2 (100 ng/ml) just before and for 4 min after adding to the flow chambers, and CCL2-treated cells were used with or without treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested and transmigrated, while the middle panel shows arrested cells as a percentage of cells rolling and the right panel shows transmigrated cells as a percentage of cells arresting. Each symbol shows data from cells from one donor, with four experiments using CCL2 and two experiments including the additional treatment with BMS22. Bars indicate means +/− SEM. p values were calculated using paired Student’s t tests, *, p<0.05; **, p<0.01; ***, p<0.001.

Journal: bioRxiv

Article Title: Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells

doi: 10.1101/2023.01.25.525561

Figure Lengend Snippet: (A) Culture supernatants collected from the upper and lower wells of transwells with HUVEC monolayers were assayed for CCL2, CCL7 and CCL8 by ELISA. Data are from three experiments and bars indicate means +/− SEM. (B) Confocal microscopy images at ×40 magnification of non-permeabilized and permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL20, CCL5, CXCL9, CCL2, CCL7 or CCL8 (green) along with nuclear staining (blue) as described in Material and methods. The scale bars indicate 10 µm. Images are representative of three experiments. (C) CCR6 + CCR2 + cells were either left untreated or treated with CCL2 (100 ng/ml) just before and for 4 min after adding to the flow chambers, and CCL2-treated cells were used with or without treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested and transmigrated, while the middle panel shows arrested cells as a percentage of cells rolling and the right panel shows transmigrated cells as a percentage of cells arresting. Each symbol shows data from cells from one donor, with four experiments using CCL2 and two experiments including the additional treatment with BMS22. Bars indicate means +/− SEM. p values were calculated using paired Student’s t tests, *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Samples were incubated with primary antibodies against chemokines CCL2 (5 μg, MAB679) CCL5 (5 μg, MAB278), CCL7 (5 μg, MAB282), CCL8 (5 μg, MAB281), CCL20 (5μg, AF360), CXCL9 (5 μg, MAB392), mouse IgG 1 isotype control (5 μg, MAB002) and mouse IgG 2B isotype control (MAB004) all from R&D Systems, at RT for 2 h. Cells were washed three times with PBS before incubating with secondary antibody Alexa Fluor 488 (1:500, A-11017; Invitrogen) for 1 h and after washing three times with PBS, were counter-stained with DAPI (1μg/ml 62248; Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining

(A) CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black and C-terminal GAG-binding sequence of CXCL9 in red (top). Confocal microscopy images of control or CCL2-CXCL9 chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and DAPI (blue) as described in Materials and methods (bottom). (B) Numbers of CCR6 + CCR2 + cells, either untreated or treated with the CCR2 antagonist, BMS22, rolling, arrested and transmigrated on TNF- α -activated HUVECs transduced with either control virus or with virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as a percentage of cells rolling and in the right panel transmigrated cells as a percentage of cells arresting. Each symbol shows data for cells from one donor, with cells from four donors in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means +/− SEM. p values were calculated using paired t tests, *, p<0.05; **, p<0.01; ***, p<0.001.

Journal: bioRxiv

Article Title: Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells

doi: 10.1101/2023.01.25.525561

Figure Lengend Snippet: (A) CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black and C-terminal GAG-binding sequence of CXCL9 in red (top). Confocal microscopy images of control or CCL2-CXCL9 chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and DAPI (blue) as described in Materials and methods (bottom). (B) Numbers of CCR6 + CCR2 + cells, either untreated or treated with the CCR2 antagonist, BMS22, rolling, arrested and transmigrated on TNF- α -activated HUVECs transduced with either control virus or with virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as a percentage of cells rolling and in the right panel transmigrated cells as a percentage of cells arresting. Each symbol shows data for cells from one donor, with cells from four donors in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means +/− SEM. p values were calculated using paired t tests, *, p<0.05; **, p<0.01; ***, p<0.001.

Techniques: Sequencing, Binding Assay, Confocal Microscopy, Control, Transduction, Virus

Inflamed endothelial cells upregulate expression of selectins and integrin ligands and secrete various chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic Th17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5 and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are diluted by blood flow to create a transendothelial gradient that induces TEM.

Journal: bioRxiv

Article Title: Chemokine positioning determines mutually exclusive roles for their receptors in extravasation of pathogenic human T cells

doi: 10.1101/2023.01.25.525561

Figure Lengend Snippet: Inflamed endothelial cells upregulate expression of selectins and integrin ligands and secrete various chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic Th17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5 and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are diluted by blood flow to create a transendothelial gradient that induces TEM.

Techniques: Expressing, Binding Assay

Review

mouse antibodies against human chemokines ccl2 (R&D Systems)

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1) Product Images from "Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines"

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