mouse antibodies against ccl2  (Bio X Cell)

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 – .

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: 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.

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: Expressing, Binding Assay

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

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

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: Irradiation, Control

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

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: Flow Cytometry, Staining

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

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: Flow Cytometry

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

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

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: Irradiation, Control

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

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: Flow Cytometry, Staining

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

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: Flow Cytometry

Journal: Brain, Behavior, & Immunity - Health

Article Title: Chemokine associations with blood cerebrospinal fluid (CSF) barrier permeability and delirium

doi: 10.1016/j.bbih.2024.100920

Figure Lengend Snippet: PCR Primer sequences . Bold/Italics denote the gene name in the revised chemokine nomenclature, while the non-bold text represents older names for these chemokines. Where no probe sequence is shown, SYBR green dye was used. For pre-made primers, assay ID is provided. Forward (F), reverse (R), probe (P).

Article Snippet: Thereafter they were rehydrated and were labelled with antibodies against CCL2 (1:200; part 840288 of DY479 (R&D Systems), IL-1β (1:50; Peprotech 500-P51), CXCL1 (1:50; R&D AF-453-NA) and CXCL10 (1:10000; Peprotech P129).

Techniques: Sequencing, SYBR Green Assay

Journal: Brain, Behavior, & Immunity - Health

Article Title: Chemokine associations with blood cerebrospinal fluid (CSF) barrier permeability and delirium

doi: 10.1016/j.bbih.2024.100920

Figure Lengend Snippet: Systemic LPS injection induced transcription of Il1b and chemokines in hippocampus and choroid plexus of mice . Graphs showing transcription of Il1b, Cxcl1, Cxcl10, Ccl2, Ccl5, Ccl11, Mip1a, Mip1b and Tarc respectively in the hippocampi (HPC) and choroid plexus (CP) of mice treated with LPS (250 μg/kg) compared to vehicle-treated mice (Vh). Fold increases in the transcripts were compared using 2-way ANOVA with region and treatment as between subjects factors. Statistically significant differences by post-hoc pairwise comparisons are denoted by ∗ (p < 0.05), ∗∗ (p < 0.01), ∗∗∗ (p < 0.001) and ∗∗∗∗ (p<0.0001). All data points are displayed in addition to the mean ± SEM for n = 7, Vh and n = 8 LPS.

Article Snippet: Thereafter they were rehydrated and were labelled with antibodies against CCL2 (1:200; part 840288 of DY479 (R&D Systems), IL-1β (1:50; Peprotech 500-P51), CXCL1 (1:50; R&D AF-453-NA) and CXCL10 (1:10000; Peprotech P129).

Techniques: Injection

Journal: Brain, Behavior, & Immunity - Health

Article Title: Chemokine associations with blood cerebrospinal fluid (CSF) barrier permeability and delirium

doi: 10.1016/j.bbih.2024.100920

Figure Lengend Snippet: Transcription of chemokines and cytokines in the hippocampus and choroid plexus 24 h following laparotomy in mice. Graphs depicting relative expression of transcripts for Il1b, Tnfa , Cxcl1 , Cxcl10, Ccl3 , Ccl2 , Ccl4 , Ccl5 , Ccl11 and Ccl17 in the hippocampus (HPC) and choroid plexus (CP) of male and female mice subjected to a surgical laparotomy, compared to those that received anaesthesia but no surgical procedure (NS). Bars represent as means ± SEM or medians ± IQR n = 13 for NS controls and n = 13 for laparotomy. To maximally exploit the factorial design, groups were analysed by 2 way ANOVA and contingent on significant main effects or interaction, Bonferroni post-hoc comparisons of NS and laparotomy were performed. ∗P < 0.05, ∗∗P < 0.001.

Article Snippet: Thereafter they were rehydrated and were labelled with antibodies against CCL2 (1:200; part 840288 of DY479 (R&D Systems), IL-1β (1:50; Peprotech 500-P51), CXCL1 (1:50; R&D AF-453-NA) and CXCL10 (1:10000; Peprotech P129).

Techniques: Expressing

Journal: Brain, Behavior, & Immunity - Health

Article Title: Chemokine associations with blood cerebrospinal fluid (CSF) barrier permeability and delirium

doi: 10.1016/j.bbih.2024.100920

Figure Lengend Snippet: IL-1β and chemokine immunohistochemistry in the choroid plexus, epithelial, endothelial and ependymal cells. C57BL6J mice were intraperitoneally challenged with LPS (100 μg/kg) and 3 h later the animals were transcardially perfused with 10% formalin and paraffin-embedded. The brains were sectioned in a microtome (10 μm) and stained for A,D) IL-1β (1:50, Peprotech 500-P51, B,E) CCL2 (1:100, R&D ELISA kit) and C,F) CXCL10 (1:10000, Peprotech P129). Photomicrographs are shown as follows: A-C) Choroid plexus epithelial and stromal cells as well as ependymal border cells and D-F) blood vessels in the hippocampus. Photographs were obtained at ×40 magnification. Scale bar = 25 μm.

Article Snippet: Thereafter they were rehydrated and were labelled with antibodies against CCL2 (1:200; part 840288 of DY479 (R&D Systems), IL-1β (1:50; Peprotech 500-P51), CXCL1 (1:50; R&D AF-453-NA) and CXCL10 (1:10000; Peprotech P129).

Techniques: Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay