neutralizing anti-ccl2 monoclonal antibody  (Thermo Fisher)

Journal: Journal of Neuroinflammation

Article Title: Sigma-1 receptor regulates HIV-1 and methamphetamine-induced endothelial/pericyte barrier impairment via strain-specific inflammatory responses and mitochondrial dysregulation

doi: 10.1186/s12974-026-03750-1

Figure Lengend Snippet: METH enhances HIV-1 NL4-3 replication in HBVPs independently of Sigma-1R. A - B Impact of METH (25 µM) on HIV-1 replication as measured by p24 antigen release levels in HBVPs infected with HIV-1 NL4-3 ( A ) or JR-CSF ( B ). Data are means ± SEM ( n = 3). C - D Impact of METH (25 µM) on HIV-1 replication as measured by HIV-1 Gag mRNA expression levels in HBVPs infected with HIV-1 NL4-3 ( C ) or JR-CSF ( D ) ( n = 4–5). E Impact of pretreatment with S1RA (10 µM) for 6 h on NL4-3 HIV-1 replication in the presence and absence of METH ( n = 3–4). F Impact of HBVP pretreatment with the CXCR4 chemokine receptor antagonist AMD070 (5 µM) for 1 h on HIV-1 NL4-3 replication in the presence and absence of METH ( n = 4). G The heat map demonstrating the impact of HIV-1 NL4-3 infection and/or METH treatment for 72 h on gene expression profile of 42 ISGs in HBVPs ( n = 6). Genes with high expression levels are represented in shades of red, while those with low expression levels are shown in shades of green. Gene names are shown on the x axis. Red arrows indicate genes that were significantly differentially regulated in the HIV-1 NL4-3 + METH group compared to the control group, as determined by the RT² Profiler PCR Array ( p < 0.05). H - K RT-qPCR analysis of mRNA expression of CCL2 ( H ), MX2 ( I ), IFI30 ( J ), and PRKD2 ( K ) in HBVPs infected with HIV-1 NL4-3 and/or treated with METH ( n = 12). L Impact of blocking endogenous CCL2 with anti-human CCL2 neutralizing antibody on p24 release in HIV-1 NL4-3-infected HBVPs, with or without METH, at 72 h post-infection ( n = 6). M Impact of pretreatment with the CXCR4 chemokine receptor antagonist AMD070 (5 µM) for 1 h on CCL2 release in the presence and absence of METH at 72 h post-infection ( n = 6). Data are means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Abbreviations as in Fig. ; CCL2 - C-C motif chemokine ligand 2

Article Snippet: To neutralize CCL2, cultures were incubated with an anti-human CCL2 neutralizing antibody (8 μg/mL; R&D Systems, Cat# AF-279-NA) or with normal goat IgG as a control (R&D Systems, Cat# AB-108-C).

Techniques: Infection, Expressing, Gene Expression, Control, Quantitative RT-PCR, Blocking Assay

Journal: Journal of Neuroinflammation

Article Title: Sigma-1 receptor regulates HIV-1 and methamphetamine-induced endothelial/pericyte barrier impairment via strain-specific inflammatory responses and mitochondrial dysregulation

doi: 10.1186/s12974-026-03750-1

Figure Lengend Snippet: Synergistic impact of METH and CXCR4-Tropic HIV-1 on pericyte-dependent endothelial barrier breakdown via CXCR4/CCL2-driven viral replication and Sigma-1R-mediated mitochondrial and inflammatory dysregulation. This proposed model depicts intersecting pathways through which CXCR4-tropic HIV-1 and METH synergistically compromise endothelial barrier integrity via viral replication, Sigma-1R-mediated mitochondrial dysfunction, and modulation of IL6-associated inflammatory response. Notably, METH-enhanced replication of CXCR4-tropic HIV-1 in pericytes appears to occur independently of the Sigma-1R signaling ( www.BioRender.com )

Article Snippet: To neutralize CCL2, cultures were incubated with an anti-human CCL2 neutralizing antibody (8 μg/mL; R&D Systems, Cat# AF-279-NA) or with normal goat IgG as a control (R&D Systems, Cat# AB-108-C).

Techniques:

Journal: Poultry Science

Article Title: Prolactin promotes transepithelial migration of lymphocytes through CCL2

doi: 10.1016/j.psj.2025.105322

Figure Lengend Snippet: (A) The co-culture setup of mammary epithelial cells and T lymphocytes. (B) The number of lymphocytes in the lower chamber was counted by a cell counter, ** P < 0.01; (C) Lymphocytes across breast epithelial cells were observed by fluorescence confocal microscopy; the left and right images are the cross-section and a vertical view of Transwell chamber, respectively. (D) The number of migrated lymphocytes under prolactin stimulation with or without CCL2 neutralization was quantified using a cell counter. CCL2 neutralizing antibody was added to the lower chamber simultaneously with prolactin, ** P < 0.01; (E) Lymphocytes across breast epithelial cells were observed by fluorescence confocal microscopy; the left and right images are the cross-section and a vertical view of the Transwell chamber, respectively.

Article Snippet: One group was treated with 50 ng/mL prolactin (1445-PR, R&D Systems) and 5 μg/mL CCL2 neutralizing antibodies (goat anti-mouse CCL2, AF-479-NA, R&D Systems) to block CCL2 ( ).

Techniques: Co-Culture Assay, Fluorescence, Confocal Microscopy, Neutralization

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: Inflammatory ICAM1 + fibroblasts expand in a murine model of ligature-induced periodontitis. (A) Flow cytometry gating strategy for analysis of lineage-negative (CD45 - CD31 - EpCam - Ter119 - ) pericytes, gingival fibroblasts and ICAM1 + fibroblasts in non-ligated control (NL) and ligature induced periodontitis (LIP) group. (B) Quantification of percent fibroblasts (Lin - PDGFRA + ) and pericytes (Lin - CD146 + ) normalized by Lin - mesenchymal cell numbers in NL and LIP groups. Each dot represents one mouse as a split-mouth design. (C) Quantification of percent ICAM1 + fibroblasts (Lin - PDGFRA + ICAM1 + ) normalized by total fibroblast numbers. Each dot represents one mouse as a split-mouth design. (D) Representative immunofluorescent images of NL and LIP paraffin sections stained with antibodies specific against ICAM1 (red) and PDGFRA (green). Arrows point to ICAM1 + PDGFRA + cells. Scale bar, 100μm; inset scale bar, 10μm. (E) Quantification of ICAM1 fibroblast numbers normalized by lamina propria area (mm 2 ) comparing NL and LIP groups from the immunofluorescence experiments. (F) Left, representative immunocytochemistry images of primary gingival fibroblasts stained with ICAM1 antibody comparing control versus stimulated groups. Lipopolysaccharide from P. gingivalis (LPS, 1 ug/ml) and tumor necrosis factor alpha (TNF, 10 ng/ml) were used for stimulation. Scale bar, 20 μm. Right, quantification of ICAM1 + fibroblast numbers normalized by total fibroblast cells comparing control and LPS+TNF group. (G) Schematic diagram of qPCR for fibroblast-derived cytokines comparing FACS-sorted ICAM1 - and ICAM1 + fibroblasts in human and mouse models of periodontitis. (H, I) Quantification of relative mRNA expression by qPCR for CXCL13, CXCL1, CXCL2, CCL19, and CCL2 comparing sorted ICAM1 - and ICAM1 + fibroblasts. (H) Gingival tissue specimens from N=8 periodontitis patients; each dot represents individual patient. (G) Gingival tissues harvested from mice with LIP; each dot represents pooled samples from 2-3 mice for a total of 4 data points (N=10 mice). Data represent mean ± SEM. Welch’s t-test (B–H) and Mann-Whitney U test (I) comparing control vs. experimental group; *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Staining, Immunofluorescence, Immunocytochemistry, Derivative Assay, Expressing, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: ICAM1 + gingival fibroblasts modulate periodontal inflammation to mitigate bone loss

doi: 10.3389/fimmu.2024.1484483

Figure Lengend Snippet: (A) Left, representative flow cytometry histogram of CCL2 signal in 7d ligated control and experimental mice. Middle and right, quantification of percent CCL2 + ICAM1 + fibroblasts (Lin - PDGFRA + ) and CCL2 + pericytes (Lin - CD146 + ) normalized by lineage-negative mesenchymal cell numbers. (B) Representative flow cytometry histogram of ICAM1 expression in fibroblasts pre-gated for CCL2 + signal in control or ICAM1 + oral fibroblast-enriched conditions (LPS + TNF) treated with or without BMS-345541 in vitro. (C) Left, quantification of ICAM1 + CCL2 + fibroblasts cell counts by flow cytometry analysis. Right, ELISA analysis of CCL2 concentration in the supernatant of cultured control or ICAM1 + enriched fibroblast conditions with or without BMS-345541. (D) Top, representative gating strategy for CCL2 + cell phenotyping by flow cytometry using tissues collected from CCL2 mCherry reporter mice that had ligature placed for 7 days. Bottom right, quantification of CCL2 + fibroblasts (CD45 - EpCAM - PDGFRA + ), leukocytes (CD45 + ), endothelial cells (CD31 + ), epithelial cells (EpCAM + ), and pericytes (CD31 - CD146 + ) normalized by total CCL2 + cells. Each dot represents one mouse (N=4). (E) Top, representative immunofluorescent images of non-ligated control (NL) and ligature induced periodontitis (LIP) from CCL2 mCherry reporter mice. Paraffin sections were stained with antibodies specific against PDGFRA (green) and red fluorescent protein (red), and immunopositivity in the lamina propria and periodontal ligament space (PDL) was examined. Scale bar, 20μm.Bottom, quantification of percent CCL2 + fibroblasts (CCL2 + PDFGRA + ) normalized by total nucleated cells in field of view. N=3, split mouth design. (F) Schematic diagram of in vitro phagocytosis assay using conditioned media from ICAM1 + enriched oral fibroblast culture and primary bone marrow-derived macrophages. (G) Top, flow cytometry gating strategy for identification of double positive F4/80 + fluorescence beads + from in vitro phagocytosis assay. Bottom, representative flow cytometry histogram of fluorescence beads signals showing three distinct peaks from control, conditioned media (CM), and CM + anti-CCL2 neutralization groups. (H) Quantification of fluorescence beads + F4/80 + macrophage numbers per 10 4 events. Left, number of beads + F4/80 + with a first peak (one bead) in the histogram; right, number of beads + F4/80 + with a second or third peak (two or three beads phagocytosed). N=3 each. All in vitro experiments were repeated independently twice. Data represents mean ± SEM. For (A) , one-way ANOVA test followed by pairwise t-test’s with Šidák’s correction was performed. For (C) Brown Forsythe ANOVA test with Dunnett’s T3 Multiple comparison test; ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: BMMs were then stimulated with low-dose LPS (10 ng/ml) for 24h to induce macrophage phagocytotic phenotype, followed by incubation in conditioned media from the ICAM1 + enriched or control fibroblasts, with or without neutralizing anti-CCL2 monoclonal antibody (20 μg/ml, eBioscience, 16-7096-81).

Techniques: Flow Cytometry, Control, Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Staining, Phagocytosis Assay, Derivative Assay, Fluorescence, Neutralization, Comparison

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Primers for qPCR

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: LILRB4 is associated with microglial inflammatory phenotypes and morphology after tMCAO. ( A ) Gene expression of M1-associated phenotype markers (MCP-1, TNF-α, IL-1β, and CD32) and M2-associated phenotype markers (Arg-1, TGF-β, and CD206). ( n = 6; ** p = 0.0061, *** p = 0.0007, **** p < 0.0001, * p = 0.0222; ns p >0.05). ( B , C ) Fluorescence imaging of microglia in the infarct border region in Control, LILRB4-KO, and LILRB4-TG mice. The lower shows Sholl analysis, where the cell body is the center, and the number of points intersecting several concentric circles is calculated. Shows the number, length of microglia processes (branches), scale bar 10 μm. ( n = 10, * p = 0.0397/0.0306/0.0285)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Gene Expression, Fluorescence, Imaging, Control

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Microglia LILRB4 deficiency increases the CCL2 production. ( A ) qPCR analysis of CCL2, CCL5, CXCL1, CXCL5, CXCL10 in Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0109). ( B ) UMAP plots of 19 cell populations identified by single-cell spatial transcriptomics analysis and the expression level of CCL2 among each cell type in mice after stroke. ( C ) Spatially transcriptome heatmaps of expression patterns of CCL2 across tissue sections from sham or stroke mouse. ( D ) qPCR analysis of CCL2 in microglia of Control and LILRB4-KO mice 1 day after tMCAO. ( n = 6; * p = 0.0229)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Control, Expressing

Journal: Journal of Neuroinflammation

Article Title: Microglia LILRB4 upregulation reduces brain damage after acute ischemic stroke by limiting CD8 + T cell recruitment

doi: 10.1186/s12974-024-03206-4

Figure Lengend Snippet: Blockade of CCL2 or addition of Arg1 suppress CD8 + T cell activation and migration in co-culture with LILRB4-KD microglia. ( A , B ) Differential expression of LILRB4 in BV2 microglia transfected by knockdown and negative control lentiviral vectors. The expression of LILRB4 in BV2 was detected by PCR ( A ) and Flow cytometry assay ( B ) ( n = 3; ** p = 0.0025/0.0065/0.0057). ( C ) Experimental procedure. Transwell-placed, Control, and LILRB4-KD microglia (without or with CCL2 inhibitor or IgG) were cultured for 4 h under OGD conditions. During reoxygenation, the t-cell-containing Transwell device was placed on a 24-well plate and exposed to medium on its lower surface for 24 h, and the levels of t-cell migration to the lower layer were measured by Flow cytometry. In another experiment, microglia cells were co-cultured with CD8 + T cells. Control and LILRB4-KD microglia were collected and cultured under OGD conditions for 4 h, and CD8 + T cells were added directly to the medium during reoxygenation. One group was added recombinant Arg-1 and another group was not. After 24 h, CD69 and IFN-γ expression in CD8 + T cells were detected by flow cytometry. ( D ) T cell migration after exposure to OGD/R, measured by the number and type of T cells microglia into 24-well plates, with or without CCL2 inhibition. Flow cytometry tests for T cell migration and ratio of CD8 + T cell. ( n = 4; * p = 0.0187/0.0383/0.0104, ** p = 0.0029). ( E ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. CD8 + T cells were collected for flow cytometry detection of CD69 and IFN-γ expression. ( F ) Quantitation and statistical evaluation of data in ( E ). ( n = 6; * p = 0.0106/0.0427, *** p = 0.0006, ** p = 0.0024/0.0024/0.0029). ( G ) Control or LILRB4-KD microglia were exposed to OGD/R and co-cultured with CD8 + T cells with or without the addition of recombinant Arg-1. Flow cytometry tests for T cell proliferation. ( n = 5; * p = 0.0348/0.0487/0.0153)

Article Snippet: Anti-CCL2 neutralizing antibodies (BE0185, Bioxcell) were added to the lower chamber, with the isotype IgG as a control.

Techniques: Activation Assay, Migration, Co-Culture Assay, Quantitative Proteomics, Transfection, Knockdown, Negative Control, Expressing, Flow Cytometry, Control, Cell Culture, Recombinant, Inhibition, Quantitation Assay