anti ccl17  (Proteintech)

Journal: Nature Cardiovascular Research

Article Title: Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis

doi: 10.1038/s44161-023-00413-9

Figure Lengend Snippet: Fig. 5 | CCL17–CCR8–CCL3 axis critically interferes with Treg differentiation. a, Schematic of co-culture experiment, where isolated splenic CD4+CD62L+ T cells from Ccr8WTApoe−/− or Ccr8KOApoe−/− mice were combined with sorted CD11c+MHCII+ cDCs from LN of CCR8WTApoe−/− mice and cultured for 3 d in the absence or presence of recombinant murine CCL17 (100 ng ml−1). b, CCL3 concentrations were measured in cell supernatants by ELISA (number of independent experiments per bar from left to right, n = 7, 9, 6, 9). c, CD4+CD25+Foxp3+ Treg cells were quantified using flow cytometry analysis (number of independent experiments per bar from left to right: n = 10, 12, 10 and 13). d, Scheme of co-culture experiment, where isolated splenic CD4+CD62L+ T cells from Ccr8WTApoe−/− or Ccr8KOApoe−/−mice were combined with sorted CD11c+MHCII+ cDCs from LN of Apoe−/− or Apoe−/−Ccl17e/e mice and cultured for 3 d. e, CCL3 concentrations in the supernatant were determined by ELISA

Article Snippet: Proximity ligation was carried out using the Duolink In Situ Red kit goat/rabbit (Sigma-Aldrich) on PFA-fixated mouse DCs cultured on collagen-coated coverslips that were pre-incubated with recombinant mouse CCL1 (Peprotech) and CCL17 (BioLegend) using primary polyclonal antibodies to mouse CCL17 (R&D systems), mouse CCL1 (Acris), mouse CCR4 (Thermo Scientific), mouse CCR5 (Santa Cruz Biotechnology) and mouse CCR8 (Abcam) according to the manufacturer’s instructions.

Techniques: Co-Culture Assay, Isolation, Cell Culture, Recombinant, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature Cardiovascular Research

Article Title: Identification of a non-canonical chemokine-receptor pathway suppressing regulatory T cells to drive atherosclerosis

doi: 10.1038/s44161-023-00413-9

Figure Lengend Snippet: Fig. 7 | CCL17-dependent CCL3 release controls Treg differentiation via CCR1. a, Experimental scheme wherein CD4+CD62L+ T cells isolated from spleens of Apoe−/−, Apoe−/−Ccr1−/− or Apoe−/−Ccr5−/− mice were cultured for 3 d under Treg-polarizing conditions (TGFβ at 100 ng ml−1) in the presence or absence of recombinant mouse CCL3 (100 ng ml−1). b, Quantification of CD45+CD4+CD25+FoxP3+ Treg cells (number of independent experiments per bar from left to right, n = 7, 7, 7, 7, 7, 7, 6, 6 and 6) using flow cytometry. c, Scheme of co-culture experiment wherein CD4+CD62L+ T cells isolated from spleens of Apoe−/−, Apoe−/−Ccr1−/− or Apoe−/−Ccr5−/− mice were combined with sorted CD45+CD11c+MHCII+eGFP+ cDCs from LNs of Apoe−/−Ccl17wt/e or Apoe−/−Ccl17e/e mice and cultured for 3 d. d, Quantification of CD45+CD4+CD25+FoxP3+ Treg cells (number of independent experiments per bar from left to right, n = 5, 5, 5, 4, 5 and 3) using flow cytometry. e, Experimental scheme of Apoe−/− or Apoe−/−Ccr1−/−

Article Snippet: Proximity ligation was carried out using the Duolink In Situ Red kit goat/rabbit (Sigma-Aldrich) on PFA-fixated mouse DCs cultured on collagen-coated coverslips that were pre-incubated with recombinant mouse CCL1 (Peprotech) and CCL17 (BioLegend) using primary polyclonal antibodies to mouse CCL17 (R&D systems), mouse CCL1 (Acris), mouse CCR4 (Thermo Scientific), mouse CCR5 (Santa Cruz Biotechnology) and mouse CCR8 (Abcam) according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Recombinant, Flow Cytometry, Co-Culture Assay

Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 1 Circulating CCL17 and CCL22 chemokine levels in morbidly obese patients and age-matched controls. (A) CCL17 and (B) CCL22 levels were measured in plasma samples from obese patients (n = 60) and controls (n = 20). Scatter dot plots showing median with interquartile range. Comparison between groups were made by Mann Whitney test. Spearman test shows a positive correlation between CCL17 and CCL22 with HOMA- IR Index (C, D) and BMI (E, F) (n = 20 control subjects and n = 60 morbidly obese patients).

Article Snippet: Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Clinical Proteomics, Comparison, MANN-WHITNEY, Control

Journal: Frontiers in endocrinology

Article Title: CCL17 and CCL22 chemokines are upregulated in human obesity and play a role in vascular dysfunction.

doi: 10.3389/fendo.2023.1154158

Figure Lengend Snippet: FIGURE 2 Expression of CCL17 and CCL22 is increased in VCAT from morbidly obese patients. Relative quantification of mRNA levels for (A) CCL17 and (B) CCL22. Comparisons between groups were made by Wilcoxon matched-pair signed-rank test. Values are expressed as mean ± SEM (n = 33). (C) CCL17 and (D) CCL22 chemokine release into conditioned media was determined after 48 h of SCAT and VCAT explant culture. Chemokine secretion is expressed as pg/ml in the supernatant. Values are expressed as mean ± SEM (n = 22). Comparison between groups were made by Mann Whitney test. (E) Immunofluorescence representative images showing colocalization of CCL17 with CD3 (lymphocytes), CD31 (endothelial cells) and Mac-3 (macrophages); or CCL22 with CD3, CD31, Mac-3 in VCAT. Immunoreactivity was visualized using Alexa Fluor 594 (CCL17 and CCL22, red) and Alexa Fluor 488 (CD31, CD3, Mac-3, green) secondary antibodies. Nuclei were stained with Hoechst (blue). Scale bar, 20 mm. Nuclei were stained with Hoechst (blue).

Article Snippet: Antigen was unmasked with proteinase K (cat#S3020, Dako, Santa Clara, CA) and blocked with 15% horse serum for 1 h. Samples were incubated with the following primary antibodies overnight at 4°C: mouse anti-human CCL17 (1:50, cat#DY364-05, R&D Systems), mouse anti-human CCL22 (1:50, cat#DY336, R&D Systems), goat anti-human CCR4 (1:100, ab1669; Abcam, Cambridge, UK), rabbit anti-human CD3 (1:100, cat#C7930, Sigma-Aldrich, St. Louis, MO), rabbit polyclonal anti-human CD31 (1:50, cat#ab32457, Abcam) and rat anti-human Mac-3 (1:100, cat#sc19991, Santa Cruz Biotechnology, Dallas, TX).

Techniques: Expressing, Comparison, MANN-WHITNEY, Staining