anti ccl2 neutralizing antibody  (R&D Systems)

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

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Cytokine expression and increased AKT activity are independent mechanisms without a tumor cell intrinsic effect on rebound growth. ( a ) Luminex-based multiplex assay results showing cytokine secretion during withdrawal after treatment for five days with either DMSO (solvent control) or 5 nM dabrafenib. Data is shown as mean ± SD ( n = 3 independent biological replicates). Two-tailed unpaired t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( b ) Kinase phosphorylation array of samples treated for five days with 5 nM dabrafenib or DMSO (solvent control) followed by 24 h withdrawal (wd). Arrays consist of two membranes, each target is detected in technical duplicates. Images shown are representatives of two biological replicates. ( c - d ) Western blot analysis of AKT phosphorylation after five days treatment with 5 nM dabrafenib (dabra) followed by up to 72 h withdrawal. Blots shown are representative of three independent biological replicates ( c ). Quantification ( d ) is relative to solvent control (DMSO; dashed line) and shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( e - f ) Western blot analysis of AKT activity markers after treatment with recombinant cytokines. Treatment for five days with DMSO or 5 nM dabrafenib (dabra) served as negative and positive control for western blots ( e ). Blots shown are representative of three biological replicates ( e ). Quantification ( f ) was done relative to untreated samples (dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test; no indication: not significant. ( g ) RT-qPCR analysis of chemokine gene expression after five days treatment with 5 nM dabrafenib (dabra) alone or in combination with varying concentrations of alpelisib (alp). Quantification was done relative to dabrafenib only treatment (0; dashed line) and is shown as mean ± SD ( n = 3 independent biological replicates). One-sample t-test, *p-value ≤ 0.05 **p-value ≤ 0.01; no indication: not significant. ( h ) Viable cell counts during treatment with 5nM dabrafenib (dabra) alone or in combination with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa) followed by ten days of withdrawal. Dashed line indicates withdrawal timepoint. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates. ( i ) Viable cell counts during treatment with 5 nM dabrafenib (dabra) followed by withdrawal. During withdrawal cells were either untreated (= solvent) or treated for five days with a combination of antibodies neutralizing CCL2 (0.5 µg/mL), CX3CL1 (0.25 µg/mL), CXCL10 (0.25 µg/mL) and CCL7 (0.1 ng/mL) (neuABs), IgG control (1 µg/mL), 5 µM alpelisib (alp) or 1 µM ipatasertib (ipa), followed by five days of withdrawal. Dashed lines indicate withdrawal timepoints. Viable cell counts are normalized to treatment start (-5d). Data is shown on a logarithmic scale (base 10) as mean ± SD of at least three biological replicates

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Expressing, Activity Assay, Luminex, Multiplex Assay, Solvent, Control, Two Tailed Test, Phospho-proteomics, Western Blot, Recombinant, Positive Control, Quantitative RT-PCR, Gene Expression

Journal: Journal of Neuro-Oncology

Article Title: Rebound growth of BRAF mutant pediatric glioma cells after MAPKi withdrawal is associated with MAPK reactivation and secretion of microglia-recruiting cytokines

doi: 10.1007/s11060-024-04672-9

Figure Lengend Snippet: Increased cytokine secretion upon MAPKi treatment and withdrawal induces increased microglia attraction. ( a ) Schematic of transwell migration assay setup to investigate migration of HMC3 cells towards conditioned media (CM) collected from BT-40. ( b ) Transwell migration assay of HMC3 cells towards conditioned media (CM) collected from BT-40 cells after 24 h of withdrawal after five days treatment with DMSO (solvent control), 5nM dabrafenib (d) or 2.7 nM dabrafenib and 0.3 nM trametinib (d + t). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test, * p-value ≤ 0.05; no indication: not significant. ( c ) Transwell migration assay of HMC3 cells towards CM collected 24 h after dabrafenib withdrawal (dabra wd) containing either antibodies neutralizing CCL2 (1 µg/mL), CX3CL1 (0.5 µg/mL), CXCL10 (0.5 µg/mL) and CCL7 (0.2 ng/mL) (neuABs) or IgG (2 µg/mL). 2% FCS serves as baseline control as CM contains 2% FCS, 0% FCS as negative control and 10% FCS as positive control. Quantification is shown as mean ± SD ( n = 3 independent biological replicates; 2 technical duplicates per condition; 10–12 randomly distributed images were quantified per transwell) relative to 2% FCS. One-sample t-test and two-tailed unpaired t-test (comparing IgG to neuABs), *p-value ≤ 0.05 ***p-value ≤ 0.001; no indication: not significant. ( d ) Representative fluorescence images showing HMC3 migrated through the transwell, nuclei were stained with DAPI. Scale bar = 50 µM. ( e - h ) RT-qPCR analysis of CX3CL1 ( e ), CXCL10 ( f ), CCL2 ( g ) and CCL7 ( h ) expression in BT-40 xenograft tumors after six days treatment with dabrafenib (dabra; 100 mg/kg, six doses, once daily) followed by three days of withdrawal (withdrawal). Samples from mice showing tumor progression (#4, #6; Fig. d) during dabrafenib treatment were excluded from the analysis. Quantification was done relative to the median of untreated samples (control). CX3CL1 was undetected in one sample (#2), CCL2 was undetected in two samples (#2, #7) and CCL7 was undetected in six samples (#2, #3, #5, #7, #8, #11), for these samples Ct values were set to 40 (max. number of cycles). Boxplots depict the median, first and third quartiles. Whiskers extend from the hinge to the largest/smallest value no further than 1.5 * IQR from the hinge (where IQR is the interquartile range). Two-tailed unpaired t-test; no indication: not significant (control: n = 3 mice, dabra: n = 4 mice, withdrawal: n = 6 mice)

Article Snippet: For cell count experiments and western blot analysis, cells were treated with antibodies neutralizing CCL2 (0.5 μg/ml; cat. no. MAB279, R&D systems), CX3CL1 (0.25 μg/ml; cat. no. MAB3652, R&D systems), CXCL10 (0.25 μg/mL; cat. no. MAB266, R&D systems) and CCL7 (0.1ng/ml; cat. no. MAB282, R&D systems) or mouse IgG (cat. no. MAB002, R&D systems).

Techniques: Transwell Migration Assay, Migration, Solvent, Control, Negative Control, Positive Control, Two Tailed Test, Fluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: Desialylation of MDSCs downregulates MDSC functional markers and cytokines at the transcript level. Suppressive MDSC-like cells were generated in vitro by coculture with A549 or A549-sia cancer cell lines as described in Fig. . CD33 + cells were isolated on Day 7 and processed for single-cell RNA sequencing (scRNAseq). A Seurat analysis of the scRNAseq dataset projected in UMAP colored by cluster. n = 4 donors per treatment group . B The dataset was subdivided into individual groups showing MDSC-like cells generated by A549 (green, left) or A549-sia (pink, right) coculture. C Stacked bar plots showing the frequency of each cluster annotated in ( A ) subclustered in A549- and A549-sia-generated MDSC-like cells. D Gene Ontology (GO) enrichment analysis of the top 10 upregulated gene sets found in Cluster 2. Dot plot showing the mean normalized enrichment score (NES) of the GO gene sets. The color coding indicates the adjusted p values, and the dot size is proportional to the gene count found in the listed pathway. E Heatmap of selected genes per patient divided into A549- and A549-sia-generated MDSC-like cells. The genes were functionally categorized into 5 groups: (i) chemokines and chemotaxis genes; (ii) MDSC and macrophage marker genes; (iii) protumor function MDSC genes; (iv) other genes; and (v) adhesion, attachment and ECM-related genes. F Gene expression of selected markers (IL1B, S100A9, S100A8, MKi67 and CCL2) as a density plot. The expression density is shown as a scale from blue (low) to yellow (high). G Expression of CCL2 was normalized to that of the donor.The data are presented as the mean ± SD. A two-tailed paired t test ( F , E ) was used. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 µg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Functional Assay, Generated, In Vitro, Isolation, RNA Sequencing Assay, Chemotaxis Assay, Marker, Expressing, Two Tailed Test

Journal: Cellular and Molecular Immunology

Article Title: Engagement of sialylated glycans with Siglec receptors on suppressive myeloid cells inhibits anticancer immunity via CCL2

doi: 10.1038/s41423-024-01142-0

Figure Lengend Snippet: CCL2 is involved in T-cell suppression via the Siglec-sialoglycan axis in suppressive myeloid cells. A MCP-1/CCL2 found in the supernatant of murine MDSC:T-cell cocultures at the endpoint of the experiment from Fig. . MDSCs were untreated, or were pretreated with sialidase or a Siglec-E blocking antibody . n = 3 donors per group . B Correlation of MCP-1/CCL2 levels measured in supernatants of murine MDSC:T-cell cocultures at the endpoint and percentage of proliferating CD8 + T cells from the same conditions from ( A ). n = 3 donors per group . C Experimental setup: Neutralization of CCL2 using a neutralization antibody in SigE ΔLysM mice and SigE WT littermates bearing B16F10 tumors. Mice were injected with a CCL2 neutralization antibody up to 3 times a week (gray arrow) starting 1 day after subcutaneous B16F10 tumor injection (black arrow). Tumor growth and survival were monitored, and the suppressive capacity of MDSCs was analyzed in vitro. D Kaplan‒Meier survival curves from pooled data from 2 independent experiments. n = 5–8 mice per group . E B16F10 tumors at the endpoint ( C ) were digested, cocultured with pervanadate and analyzed by phospho-flow cytometry for phosphorylated STAT3 (pSTAT3). The percentage of pSTAT3 gated on total MDSCs is shown. Representative histograms for each condition are shown on the right. n = 3 mice per group . F Suppressive capacity of MDSCs against naïve T cells. Percentage of proliferating CD8 + T cells cocultured without MDSCs, ( G ) with MDSCs from SigE WT mice or ( H ) MDSCs from SigE ΔLysM mice with or without addition of CCL2 blocking antibody. N = 3–5 mice from N = 3 experiments . I Cytokine expression found in the supernatant of human primary CD33 + :CD8 + cell cocultures at the endpoint of the experiment from Fig. . CD33 + cells were left untreated or pretreated with sialidase. Z scores were calculated for each cytokine and are shown on a color scale from blue (low) to red (high). n = 3 donors per group . The data are presented as mean ± SD. Two-tailed paired t tests or unpaired t tests ( E ) were used. R shows the Pearson correlation coefficient. For survival analysis, the log-rank test was used, followed by the Šidák correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001

Article Snippet: For neutralization of CCL2 in vivo, mice were injected intraperitoneally 3 times a week with 200 µg/mouse of an anti-CCL2 neutralizing antibody (clone: 2H5, BioXCell) in PBS.

Techniques: Blocking Assay, Neutralization, Injection, In Vitro, Flow Cytometry, Expressing, Two Tailed Test