Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. Uniform manifold approximation and projection (UMAP) projection of single cells clustered with identification of macrophage identity. Identification of cardiac resident macrophages (CRMs) and monocyte-derived macrophages (MoMFs) based on the expression of marker genes Folr2 (Folate receptor beta), Timd4 (T cell immunoglobulin and mucin domain containing 4), Lyve1 (lymphatic vessel endothelial hyaluronan receptor 1), Il1β (Interleukin 1 beta), H2-Ab1 (Histocompatibility 2, class II antigen A, beta 1), and Ccr2 (C-C chemokine receptor type 2) are shown. B. Sham and transverse aortic constriction (TAC)–derived cells are plotted separately to visualize CCL24 abundance in both conditions. C. Heatmap of differentially expressed genes (DEGs) between CCL24+ and CCL24- macrophages. The identity of both subsets is indicated by top DEGs in sham and TAC conditions. D. CCL24 measured from whole cardiac lysates of sham- and TAC-operated hearts collected 1- or 4 weeks post-TAC. (n = 6, 6, 3, 5). E. IL-4 and IL-10 from sham- and TAC-operated hearts collected 1-week post-TAC (n = 3, 2, 2, 6). F. CCL24 secreted by isolated cardiac mϕs stimulated with 10ng/ml IL-4, IL-10 or both for 48 hours. (n = 3, 2, 2, 6). G. CCL24 measured from whole cardiac lysates of WT or IL4 knockout (IL4KO) TAC-operated mice collected 1-week post-surgery (n = 5,4). H. Flow cytometry analysis of IL-4 receptor on the surface of residential and recruited macrophages with gating strategy (left), mean fluorescence intensity (MFI) histograms (middle), and quantification (right) (n=5). I. Time course of systolic cardiac function at baseline, 1-, 2- and 4-weeks post TAC. EF – Ejection Fraction, FS – Fractional Shortening, CO – Cardiac Output. (Left – quantification; Right – representative M-mode echocardiography traces) (p value < 0.05 #- WT Sham vs WT TAC; *-WT TAC vs CCL24 KO TAC; + KO Sham vs KO TAC) (WT Sham: n = 11, 3, 5, 5; KO Sham: n = 11, 4, 6, 5; WT TAC: n = 8, 14, 11; KO TAC: n = 18, 15, 5). J. Schematic showing the timing of vehicle or CCL24 neutralizing antibody administration relative to transverse aortic constriction (TAC) surgery (left) and systolic cardiac function measured 1- week post-TAC following vehicle or CCL24 neutralizing antibody injections (right, n = 4, 21). Data pooled from 3 independent experiments.

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: Derivative Assay, Expressing, Marker, Isolation, Knock-Out, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. Time course of chamber measurements at baseline, 1-, 2- and 4-weeks post TAC. LVPWd – Left Ventricular Posterior Wall in diastole, LVIDd – Left Ventricular Internal Diameter in diastole (p value < 0.05 *-WT TAC vs CCL24 KO TAC; + KO Sham vs KO TAC) (WT Sham: n = 11, 3, 5, 5; KO Sham: n = 11, 4, 6, 5; WT TAC: n = 8, 14, 11; KO TAC: n = 18, 15, 5) B. Heart weight to body weight ratios (HW/BW) measured at 1- and 4-weeks post-TAC. (n = 21, 41, 21, 41). Data pooled from 3 independent experiments. C. Representative histological images (left) of WGA (wheat germ agglutinin; green) and quantification (right) of cross-sectional myocyte area from WT or CCL24 KO hearts 1-week post- TAC. D. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) for atrial natriuretic factor (Nppa), brain natriuretic peptide (Nppb), sarcoplasmic/endoplasmic reticulum Ca2+- ATPase (Serca) and Calcium/Calmodulin Dependent Protein Kinase II Delta (Camk2d) on cDNA isolated from whole cardiac lysates from WT or CCL24 KO TAC-operated mice ( D ) or Human fetal ventricular cardiomyocyte, RL-14 cell line stimulated with 100ng/mL CCL24 for 48 hours ( E ). F. Representative images (left) of WGA staining of RL14 cell line (top) and bright field imaging of neonatal rat ventricular myocytes (NRVM) (bottom) stimulated with 100ng/mL of Bovine serum albumin (BSA) or CCL24 for 48 hours. Quantification of relative myocyte size normalized to a control group (right).

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Staining, Imaging, Control

Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. 2-D Principal component analysis (PCA) of bulk RNA-sequencing from WT Sham (n = 2), CCL24 KO Sham (n = 2), WT TAC (n = 6), and CCL24 KO TAC (n = 4) groups 1-week post sham or TAC surgeries. (B-C). Top upregulated and downregulated gene sets detected by gene set enrichment analysis (GSEA) performed on differentially expressed genes between WT and CCL24 KO groups post sham (n = 333 genes) or TAC (n = 357 genes) surgeries ( B ). Enrichment plot for differentially regulated fibrotic pathways ( C, left ) and genes associated with the enrichment ( C, right ). D. Reactome pathway analysis for top upregulated and downregulated pathways between WT and CCL24 KO mice 1-week post-TAC. E. Volcano plot highlighting curated list of fibrotic genes differentially regulated in WT vs CCL24 KO mice in sham (left) and TAC (right) conditions. F. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) for type I collagen (Col1), type III collagen (Col3), type V collagen (Col5), smooth muscle alpha (α)-2 actin (Acta2), matrix metallopeptidase 2 (Mmp2), and Transforming growth factor β (Tgfβ) on cDNA isolated from whole cardiac lysates from WT or CCL24 KO TAC-operated mice. G. Representative images (left) of Movat’s Pentachrome sections derived from WT or CCL24 KO mice post-TAC surgery. The bar graph (right) shows quantification of fibrosis (yellow staining, n=13, 13). Data pooled from 3 independent experiments. (H-I). Flow cytometry on primary mouse fibroblasts isolated from hearts and stimulated with 100ng/mL CCL24 for 24 hours. Quantification of Ki67+ fibroblasts (left) and representative plots of Ki67 vs. alpha-smooth muscle actin (aSMA) staining ( H, right ). Quantification of aSMA+ fibroblast ( I, left ) and mean fluorescence intensity (MFI) histogram of aSMA ( I, right ). J. ELISA for TGFb secreted by primary mouse cardiac fibroblasts stimulated with 100ng/mL of CCL24 for 48 hours.

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: RNA Sequencing, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Derivative Assay, Staining, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. Representative flow cytometry plots of cardiac non-myocytes showing the expression of CCR3 in the cardiac fibroblasts (CFBs), endothelial and immune cells from CCR3 isotype controls, sham or TAC-operated hearts (top). Quantification of CCR3+ proportions and cell counts in non- myocyte cell compartments (bottom, n = 4, 6). B. Representative images of fibroblasts stained for collagen-1 (white), periostin (red), αSMA (alpha-smooth muscle actin) (green), and DAPI (4’,6-diamidino-2-phenylindole) (blue) following treatment with 100 ng/mL of CCL24 alone or in combination with 0.5ug/mL of CCR3 or TGFb blocking antibody (n=4). Bar graphs show quantification of collagen-1, periostin and aSMA intensity, and aSMA+ cell percentage. C. Schematic showing the timing of vehicle or CCR3 antagonist administration relative to sham or transverse aortic constriction (TAC) surgery (left). Quantification of systolic function by ejection fraction (EF) in sham or TAC-operated mice receiving either vehicle or CCR3 antagonist (n = 3,3,5,4; right). D. Strategy for producing mutants with conditional deletion of CCR3 in fibroblasts. Mice homozygous for the CCR3 floxed allele were mated with transgenic mice expressing tamoxifen- inducible Cre recombinase under the control of a platelet-derived growth factor receptor alpha (PDGFRa) promoter, resulting in deletion of CCR3 in PDGFRa+ fibroblasts (left). The bar graph represents frequency and counts of CCR3+ fibroblasts and endothelial cells in PDGF Cre CCR3 wt/wt vs PDGF Cre CCR3 fl/fl mice 1-week post-TAC surgery. E. Heart weight to body weight (HW/BW) and lung weight to body weight (LW/BW) ratios in in PDGF Cre CCR3 wt/wt vs PDGF Cre CCR3 fl/fl mice 1-week post-TAC surgery (n = 5,5). F. Systolic cardiac function from M-mode short axis echocardiography of PDGF Cre CCR3 wt/wt vs PDGF Cre CCR3 fl/fl mice 1-week post-TAC surgery (n = 4, 6). EF – ejection fraction, FS – fractional shortening.

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: Flow Cytometry, Expressing, Staining, Blocking Assay, Transgenic Assay, Control, Derivative Assay

Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. Cytometry by time-of-flight–based visualized stochastic neighbor embedding (ViSNE) plot showing the abundance of different immune cells and colored expression of CD3e (T cells), NKp46 (NK cells), B220 (B cells), Ly6G (PMNs), SiglecF (Eosinophils) and CD64 (Macrophages) in arbitrary units (AU) between WT-sham, WT-TAC, CCL24 KO-sham and CCL24 KO-TAC groups 1-week post-TAC. B. Quantification of immune cell frequency (left) and abundance (right) after TAC surgery in WT or CCL24 KO mice (n=11, 10). C. Representative flow cytometry plots (left) and quantification (right) of cardiac eosinophils (Eos), neutrophils (PMNs) and Macrophages (Macs) along with CCR2+ and CCR2- macrophages 1 week after TAC surgery in WT or CCL24 KO mice (n=12, 9). D. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) for Tumor necrosis factor alpha (TNFa), Interleukin 1 beta (IL1b) and Interleukin 6 (IL6) on cDNA isolated from whole cardiac lysates from WT or CCL24 KO TAC-operated mice (n = 7, 5).

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: Cytometry, Expressing, Flow Cytometry, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

Journal: bioRxiv

Article Title: Macrophage-derived CCL24 promotes fibrosis and worsens cardiac dysfunction during heart failure

doi: 10.1101/2024.07.12.603277

Figure Lengend Snippet: A. Representative E and A peaks from 4-chamber apical view echocardiography (left) and quantification of systolic function by ejection fraction (EF), fractional shortening (FS), and diastolic function measured by E/A peak ratio in wildtype young and old mice (right, n = 6, 7). B. Chamber dimension by left ventricular internal diameter in diastole (LVIDd) measured in wildtype young and old mice (right, n = 6, 7). C. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) for type I collagen (Col1a1), smooth muscle alpha (α)-2 actin (Acta2), matrix metallopeptidase 2 (Mmp2), and Transforming growth factor β (Tgfβ) on cDNA isolated from whole cardiac lysates from young or old mice (n = 6, 7). (D-E). Enzyme-linked immunosorbent assay (ELISA) for CCL24 measured from whole cardiac lysates of wildtype young and old hearts ( D , n = 3, 6) or isolated cardiac macrophages from wildtype young or old hearts stimulated with 10ng/mL IL-4/10 for 48 hours ( E , n = 4, 4). F. Representative flow cytometry plots (left) and quantification (right) of residential (CCR2- Folr2+) and recruited (CCR2+ Folr2-) macrophages in wildtype young vs. old hearts (n = 13,15) Data pooled from two independent experiments. G. Heart weight to body weight ratios (HW/BW) measured in aged WT vs CCL24 KO hearts. (n =7,8) H. Representative histological images (left) of WGA (wheat germ agglutinin; green) and quantification (right) of cross-sectional myocyte area (n =7,8) I. Representative E and A peaks from 4-chamber apical view echocardiography (left) and quantification of systolic function by ejection fraction (EF), fractional shortening (FS), and diastolic function measured by E/e’, global longitudinal strain (GLS) and E/A peak ratio in aged WT and CCL24 KO mice. J. Chamber dimension by left ventricular internal diameter in diastole (LVIDd) measured in aged WT and CCL24 KO mice. K. Representative images (left) of Sirius red fast green (SRFG) sections derived from aged WT or CCL24 KO mice. The bar graph (right) shows the quantification of fibrosis (n=7, 8).

Article Snippet: To block CCL24 in vivo, we administered 3 doses (100 uL) of either vehicle control or 50 ug of CCL24 neutralizing antibody (MAB528, R&D systems) via intraperitoneal injection before TAC and during the first week of recovery.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Derivative Assay

Journal: International immunology

Article Title: IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration.

doi: 10.1093/intimm/dxu090

Figure Lengend Snippet: Fig. 4. IL-10 enhanced CCL24 and Arg1 expression in IL-4-stimulated BMDMs and macrophages from different sources. BMDMs and mac- rophages from different sources were stimulated with IL-4 (10 ng ml−1), IL-10 (10 ng ml−1) and IL-4 + IL-10 to measure gene expression for 24 h and CCL24 production for 72 h. Gene expression of the M2a macrophage markers CCL24 (A, C, D) and Arg1 (E, F) in M-CSF- and GM-CSF- stimulated BMDMs (A, E); thioglycolate- and zymosan-elicited peritoneal macrophages (shown as Thio and Zymo) (C, F) and naive peritoneal macrophages (D) was determined using real-time PCR. CCL24 in culture supernatant from M-CSF-induced BMDMs was determined using ELISA (B). Data are presented as the mean ± SEM (n = 3–4). Statistical analyses were conducted between IL-4- and IL-4 + IL-10-stimulated macrophages. Statistically significant differences: *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001.

Article Snippet: Mouse CCL24 was measured using commercially available ELISA kits (DY528, R&D Systems).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: International immunology

Article Title: IL-10 enhances the phenotype of M2 macrophages induced by IL-4 and confers the ability to increase eosinophil migration.

doi: 10.1093/intimm/dxu090

Figure Lengend Snippet: Fig. 5. IL-4 + IL-10-stimulated macrophages increased eosinophil migration in vitro. Eosinophils were isolated through negative selection of Thy1.2 and B220 MACS from peritoneal fluids in a mouse model of ragweed-induced peritonitis. Eosinophils were added into the upper side of a chemotaxis chamber. CCL24 (A), culture supernatant of BMDMs stimulated for 72 h with IL-4, IL-10 or IL-4 + IL-10 (B), culture supernatant of BMDMs stimulated for 72 h with 10 ng ml−1 of IL-4, IL-10 or IL-4 + IL-10 with or without isotype control or anti-CCL24 antibody (C) or only cytokine (10 ng ml−1 of IL-4, IL-10 or IL-4 + IL-10) (D) was added to the lower side of the chamber. After 1 h incubation, migrated eosinophils were quanti- fied using the Celltiter Glo Luminescent Cell Viability Assay. The y-Axis shows relative luminescence unit (RLU), which reflects the number of live cells and is used as an index of the number of eosinophils. Data are presented as the mean ± SEM (n = 3–4). Statistically significant differences compared with control: *P ≤ 0.05 and **P ≤ 0.01.

Article Snippet: Mouse CCL24 was measured using commercially available ELISA kits (DY528, R&D Systems).

Techniques: Migration, In Vitro, Isolation, Selection, Chemotaxis Assay, Control, Incubation, Cell Viability Assay

Journal: Cell reports

Article Title: Innate Lymphoid Cells Play a Pathogenic Role in Pericarditis

doi: 10.1016/j.celrep.2020.02.040

Figure Lengend Snippet: (A) Expression of the Ccl11 gene encoding eotaxin-1 in heart homogenates was analyzed by qPCR. (B) Schematic description of cardiac fibroblasts co-culture with ILC2s separated by 0.4-μm transwell. (C) Expression of Ccl11 in cardiac fibroblasts was analyzed by qPCR. (D) Eotaxin-1 concentrations in cell culture supernatants were measured by ELISA. IL-2 and IL-7 were included in culture media and IL-33 was added where indicated (C and D). (E–H) Expression of genes in heart homogenates from WT, Rag2 −/− , and Rag2 −/− Il2rg −/− mice treated with IL-33 was analyzed by qPCR. Ccl11 (E), Ccl24 (F), Il5 (G), and Il13 (H). (I) Immunohistochemistry staining for CCL11 of heart sections from WT mice treated with PBS or IL-33. Bars: 50 μm. Data are representative of two independent experiments and displayed as the mean with SD. Unpaired t test (A) or one-way ANOVA followed by Tukey’s post hoc test (C–H) was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001. See also .

Article Snippet: After deparaffinization, heat-induced antigen retrieval and blocking, sections were stained with 5 μg/ml Polyclonal Goat Anti-Mouse CCL11 antibody (R&D Systems) and Anti-Goat HRP-DAB Cell & Tissue Staining Kit (R&D Systems) and counter stained with Hematoxylin.

Techniques: Expressing, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: Innate Lymphoid Cells Play a Pathogenic Role in Pericarditis

doi: 10.1016/j.celrep.2020.02.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After deparaffinization, heat-induced antigen retrieval and blocking, sections were stained with 5 μg/ml Polyclonal Goat Anti-Mouse CCL11 antibody (R&D Systems) and Anti-Goat HRP-DAB Cell & Tissue Staining Kit (R&D Systems) and counter stained with Hematoxylin.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 3. CCL11 induces mouse SMC chemotaxis. A, Mouse CCL11 in 0.3% FCS at the concentrations indicated (ng/mL) were placed in the bottom wells of a modified Boyden Cham- ber. 104 SMCs were placed in the top wells. The chamber was incubated for 6 hours at 37°C and then analyzed for migration as described in Materials and Methods. For comparison, recom- binant PDGF-BB (1 ng/mL) or 10% FBS were also used as che- moattractants. 0.3% serum was used as negative control. *P0.005 compared with 0 CCL11. B, Checkerboard analysis in which CCL11 (C; 100 ng/mL) was placed in the bottom, top, or both wells as indicated. *P0.005 compared with 0.3% FCS (–). C, SMC were incubated with antibodies against CCR3 (anti- CCR3), CCR2 (anti-CCR2) (1 g/mL), or irrelevant IgG for 30 minutes at 37°C before being placed in the Boyden chamber. Results are expressed as SMC/hpf SEM and represent tripli- cate experiments *P0.005 compared with 0.3% FCS. #P0.05 compared with 0.3% FCS.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Chemotaxis Assay, Incubation, Migration, Comparison, Negative Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 5. CCL11 does not modulate SMC proliferation. SMCs were plated in 35-mm wells and treated with CCL11 (10 ng/mL or 100 ng.ml) PDGF (1 ng/mL), CCL11 (100 ng/mL) PDGF (1 ng/mL), 10% FCS, or 0.3% FCS. Duplicate plates were counted on the days indicated. The medium, with fresh growth factors, was replaced every 48 hours. Results are expressed as cell number SEM and represent the average of duplicate experiments. Figure 4. CCL11 induces mouse SMC migration. SMCs were grown to confluence and then incubated in DMEM 0.1% BSA for 24 hours. Linear wounds were made in each plate and SMCs were then incubated with DMEM 0.1% BSA alone (con- trol) or in the presence of CCL11 (concentration in ng/mL indi- cated on the X axis). In some experiments, SMCs were preincu- bated for 1 hour with anti-CCR3 (CCR3 Ab) or an irrelevant antibody (IgG) before wounding and treatment with 10 ng/mL CCRL11. The wound area was measured immediately after wounding and at 24 hours. Migration is expressed as the % decrease in wound area in 24 hours compared with control. Experiments were done in duplicate, with 3 to 5 plates used for each condition. *P0.001, 10 ng/mL CCL11 compared with control; **P0.001, 10 ng/mL eotaxin CCR3 Ab compared with 10 ng/mL eotaxin alone; #NS, 10 ng/mL eotaxin IgG compared with 10 ng/mL eotaxin alone.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Migration, Incubation, Concentration Assay, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 6. Expression of CCR3 antigen in injured mouse femoral arteries. Samples were obtained from a previous study in which C57Bl/6 mice had undergone wire-induced femoral arterial injury.39 Sections from uninjured arteries (A, B), arteries harvested 5 days (C, D), 7 days (E, F), and 28 days after injury (G through J) were stained with antibodies to CCR 3 (left) or CCL11 (right). Magnifica- tion40 for A, B, E, F, G, and H and 200 for C, D, I, and J. Sections are representative of studies done on 3 different animals.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Expressing, Staining

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in eotaxin expression during 3T3-L1 adipocyte differentiation. (a) 3T3-L1 adipocytes were collected every other day during differentiation, and the relative amount of eotaxin mRNA was detected using real-time RT-PCR. (b) 3T3-L1 adipocyte supernatants were collected every other day during differentiation, and the eotaxin secretion level was detected with an ELISA kit (D0: preadipocytes; D2-D10: 2-10 days after inducing differentiation; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Change in eotaxin expression in 3T3-L1 preadipocytes due to differentiation components. Preadipocytes were treated with several different differentiation media for 24 hours. (a) The relative amount of eotaxin mRNA was determined by real-time RT-PCR, and (b) eotaxin secretion was detected with an ELISA kit (fetal bovine serum [FBS]: DMEM + 10% FBS, FBS + insulin [Ins]: DMEM + 10% FBS + Ins 1 µg/ml, FBS + IBMX: DMEM + 10% FBS + IBMX 0.5mM, FBS + dexamethasone [DEX]: DMEM + 10% FBS + Dex 0.25 mM, DM: DMEM + 10% FBS + Ins 1µg/ml + IBMX 0.5 mM + Dex 0.25 mM; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in eotaxin expression in 3T3-L1 preadipocytes and adipocytes after stimulation with TNF-α. (a) At day 8 after inducing differentiation, a time course treatment with 10 ng/ml TNF-α was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, the relative amount of eotaxin mRNA was detected by real-time RT-PCR, and (b) eotaxin secretion were detected with an ELISA kit. (c) Time-course treatment with 10 ng/ml TNF-α on preadipocytes was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and (d) eotaxin secretion level was detected with an ELISA kit ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in eotaxin expression in differentiated 3T3-L1 adipocytes following treatment with recombinant eotaxin. At 8 days after inducing differentiation, adipocytes were treated with 10 ng/ml recombinant eotaxin, and the cells were collected in a time course. The relative amount of eotaxin mRNA expression is shown ( ** P < 0.01).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Recombinant

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in eotaxin expression in 3T3-L1 adipocytes following interleukin (IL)-4 and IL-5 treatment. At day 8 after inducing differentiation, a time course treatment of 10 ng/ml (a) IL-4 and (b) IL-5 was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and the relative amount of eotaxin mRNA was detected by real-time RT-PCR. (c) At day 8 after inducing differentiation, 10 ng/ml IL-4 and (d) IL-5 were added for 24 hours, and eotaxin secretion levels were detected with an ELISA kit ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in eotaxin expression in 3T3-L1 adipocytes following leptin treatment. (a) At day 8 after inducing differentiation, a time course treatment of 100 pg/ml leptin was conducted for 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours, and the relative amount of eotaxin mRNA was detected by real-time RT-PCR. (b) At day 8 after inducing differentiation, 100 pg/ml leptin was introduced for 4 hours, 8 hours, 12 hours, and 24 hours, and eotaxin secretion levels were detected with an ELISA kit ( * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in adipose tissue eotaxin expression in different locations following diet-induced obesity and weight loss. The relative amount of eotaxin mRNA expression in (a) epididymal adipose tissues, and (b) visceral adipose tissues in the control (CON), HFD, and HFD→ND were observed by real-time RT-PCR (ND: normal diet; HFD: high-fat diet; HFD→ND: diet induced obesity and then induced weight loss by normal diet; * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Nutrition Research and Practice

Article Title: Expression of eotaxin in 3T3-L1 adipocytes and the effects of weight loss in high-fat diet induced obese mice

doi: 10.4162/nrp.2011.5.1.11

Figure Lengend Snippet: Changes in serum and bronchoalvelolar lavage fluid (BALF) eotaxin levels in the control (CON), high-fat diet fed (HFD), and high-fat diet switched to a normal diet after 26 weeks (HFD→ND) groups. (a) eotaxin levels in mice serum and (b) in BALF were detected with an ELISA kit ( * P < 0.05).

Article Snippet: Mouse eotaxin ELISA kits were purchased from R & D Systems (Minneapolis, MN, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay