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human anti ccl22 duoset elisa (R&D Systems)

Name: human anti ccl22 duoset elisa
Brand: R&D Systems
Rating: 93 (44 reviews)

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R&D Systems human anti ccl22 duoset elisa
Human Anti Ccl22 Duoset Elisa, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 44 article reviews

human anti ccl22 duoset elisa - by Bioz Stars, 2026-02

93/100 stars

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Journal: Translational Oncology

Article Title: CCL22 as an independent prognostic factor in endometrial cancer patients

doi: 10.1016/j.tranon.2024.102116

Figure Lengend Snippet: Representative images of CCL22-expression in EC: (A) intermediate expression in glandular cells (IRS=2.67), S/M (IRS=0.67); (B) high expression in glandular cells (IRS=12.00), S/M (IRS=5.33); (C) strongly positive cells in tumor distant S/M; (D) significant correlation between CCL22 in tumor- and stroma-cells; (E) CCL22 levels in tumor cells were higher than in S/M. Objective 20x, Scale bar 100 µm.

Article Snippet: Paraffin-embedded TMA of EC-patients and tissue of the control group were incubated with the polyclonal rabbit anti-human MDC (CCL22) antibody (500-P107 1:300, Peprotech) using ZytoChem Plus HRP Polymer System mouse/rabbit following the manufacturer's instructions.

Techniques: Expressing

Representative images of CCL22-expression in endometrial control with negative (A) and strong staining in glandular epithelial cells (B) and strongly positive cells in myometrium (C). Elevated CCL22-expression in glandular epithelial cells compared to stroma cells (D). Significantly lower CCL22-IRS EC compared to benign endometrium (E). Concerning CCL22 in S/M a trend to higher levels was found in the specimens of EC patients (F). Objective 20x, Scale bar 100 µm.

Journal: Translational Oncology

Article Title: CCL22 as an independent prognostic factor in endometrial cancer patients

doi: 10.1016/j.tranon.2024.102116

Figure Lengend Snippet: Representative images of CCL22-expression in endometrial control with negative (A) and strong staining in glandular epithelial cells (B) and strongly positive cells in myometrium (C). Elevated CCL22-expression in glandular epithelial cells compared to stroma cells (D). Significantly lower CCL22-IRS EC compared to benign endometrium (E). Concerning CCL22 in S/M a trend to higher levels was found in the specimens of EC patients (F). Objective 20x, Scale bar 100 µm.

Techniques: Expressing, Control, Staining

CCL22-expression in S/M increases with higher grade in EC (A). High CCL22-expression in S/M in EC is associated to poorer OS (B), but not to PFS (C). (D) High CCL22-expression in tumor epithelium is associated to prolonged OS, but not to PFS (E).

Journal: Translational Oncology

Article Title: CCL22 as an independent prognostic factor in endometrial cancer patients

doi: 10.1016/j.tranon.2024.102116

Figure Lengend Snippet: CCL22-expression in S/M increases with higher grade in EC (A). High CCL22-expression in S/M in EC is associated to poorer OS (B), but not to PFS (C). (D) High CCL22-expression in tumor epithelium is associated to prolonged OS, but not to PFS (E).

Techniques: Expressing

Isolated CCL22+ cells were identified as mainly M1-macrophages in distant myometrial tissue areas by double immunofluorescence: (A) Representative EC stained for CCL22 (red) and CD68, CD80, CD163, and DEC205 (green); (B) Proportion of CCL22+ cells, that expressed also one of those immune cells markers is presented; (C) Proportion of total expression of all markers: dominant occurrence of CD68+ and CD80+ cells; (D) Kaplan-Meier Curve for OS and PFS (E). Correlation analysis of FoxP3 and CCL22 in S/M (F), distant strongly positive cells (G), and in tumor cells (H).

Journal: Translational Oncology

Article Title: CCL22 as an independent prognostic factor in endometrial cancer patients

doi: 10.1016/j.tranon.2024.102116

Figure Lengend Snippet: Isolated CCL22+ cells were identified as mainly M1-macrophages in distant myometrial tissue areas by double immunofluorescence: (A) Representative EC stained for CCL22 (red) and CD68, CD80, CD163, and DEC205 (green); (B) Proportion of CCL22+ cells, that expressed also one of those immune cells markers is presented; (C) Proportion of total expression of all markers: dominant occurrence of CD68+ and CD80+ cells; (D) Kaplan-Meier Curve for OS and PFS (E). Correlation analysis of FoxP3 and CCL22 in S/M (F), distant strongly positive cells (G), and in tumor cells (H).

Techniques: Isolation, Immunofluorescence, Staining, Expressing

High CCL22 secretion by freshly isolated PBMCs in contrast to EC cell lines without stimulation (A). Significantly increased CCL22 in SN after coculture of PBMCs and EC cell lines (B). The addition of tumor-SN to PBMCs led to a significant increase of CCL22 levels (C), while incubation of tumor cells with PBMC-SN resulted in a decrease in CCL22 levels (D). mRNA levels of tumor cells after coculture revealed a significant increase in CCL22 of Ishikawa+ with PBMCs (E). CCL22 levels of tumor RIPA lysates also revealed a significant increase after coculture (F). All experiments were carried out in technical triplicate and repeated three times with PBMCs from different blood donors.

Journal: Translational Oncology

Article Title: CCL22 as an independent prognostic factor in endometrial cancer patients

doi: 10.1016/j.tranon.2024.102116

Figure Lengend Snippet: High CCL22 secretion by freshly isolated PBMCs in contrast to EC cell lines without stimulation (A). Significantly increased CCL22 in SN after coculture of PBMCs and EC cell lines (B). The addition of tumor-SN to PBMCs led to a significant increase of CCL22 levels (C), while incubation of tumor cells with PBMC-SN resulted in a decrease in CCL22 levels (D). mRNA levels of tumor cells after coculture revealed a significant increase in CCL22 of Ishikawa+ with PBMCs (E). CCL22 levels of tumor RIPA lysates also revealed a significant increase after coculture (F). All experiments were carried out in technical triplicate and repeated three times with PBMCs from different blood donors.

Techniques: Isolation, Incubation

( A ) GO analysis of macrophage response to LNR. ( B ) Heatmaps of macrophage-secreted chemokines in the iWAT from mice receiving sham or LNR at 6°C for 7 days ( n = 4 per group). Red and blue represent the fold increase and decrease in a gene, respectively (see color scale). ( C ) mRNA expression of macrophage-secreted chemokines in iWAT from mice described in (B) ( n = 8 per group). ( D ) Ccl22 mRNA expression in iWAT from mice described in (B) at 23° or 6°C for 7 days ( n = 6 per group). ( E ) Ccl22 mRNA expression in iWAT from mice described in (B) exposed to 6°C for different hours ( n = 6). ( F ) Serum CCL22 levels from mice described in (B) exposed to 6°C for different days ( n = 6). ( G ) Serum CCL22 levels from mice described in (B) ( n = 6 per group). ( H ) Ccl22 mRNA expression in iWAT SVF cells or mature adipocytes (MAs). ( I ) Ccl22 mRNA expression in M0, M1, and M2 macrophages derived from 10-week-old C57BL/6 bone marrows ( n = 8 per group). ( J and K ) mRNA expression (J) and IF (K) of UCP1 in beige adipocytes ( n = 6 per group). Scale bars, 25 μm. ( L to N ) Immunofluorescence of UCP1 (L), mRNA expression of thermogenic genes (M), and immunoblots of UCP1 (N) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. ( O to R ) Immunoblots and quantification of UCP1 [(O) and (P)], mRNA expression (Q) of thermogenic genes, and IF of UCP1 and H&E staining (R) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(C), (D), (G) to (J), (M), (P), and (Q)] * P ≤ 0.05, ** P < 0.01, and *** P < 0.001 by nonpaired Student’s t test. [(E) and (F)] * P ≤ 0.05 and ** P < 0.01 nonpaired Student’s t test compared with before cold stimulation.

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A ) GO analysis of macrophage response to LNR. ( B ) Heatmaps of macrophage-secreted chemokines in the iWAT from mice receiving sham or LNR at 6°C for 7 days ( n = 4 per group). Red and blue represent the fold increase and decrease in a gene, respectively (see color scale). ( C ) mRNA expression of macrophage-secreted chemokines in iWAT from mice described in (B) ( n = 8 per group). ( D ) Ccl22 mRNA expression in iWAT from mice described in (B) at 23° or 6°C for 7 days ( n = 6 per group). ( E ) Ccl22 mRNA expression in iWAT from mice described in (B) exposed to 6°C for different hours ( n = 6). ( F ) Serum CCL22 levels from mice described in (B) exposed to 6°C for different days ( n = 6). ( G ) Serum CCL22 levels from mice described in (B) ( n = 6 per group). ( H ) Ccl22 mRNA expression in iWAT SVF cells or mature adipocytes (MAs). ( I ) Ccl22 mRNA expression in M0, M1, and M2 macrophages derived from 10-week-old C57BL/6 bone marrows ( n = 8 per group). ( J and K ) mRNA expression (J) and IF (K) of UCP1 in beige adipocytes ( n = 6 per group). Scale bars, 25 μm. ( L to N ) Immunofluorescence of UCP1 (L), mRNA expression of thermogenic genes (M), and immunoblots of UCP1 (N) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. ( O to R ) Immunoblots and quantification of UCP1 [(O) and (P)], mRNA expression (Q) of thermogenic genes, and IF of UCP1 and H&E staining (R) in iWAT ( n = 3 to 6 per group). Scale bars, 50 μm. Data information: Results are presented as means ± SEM. [(C), (D), (G) to (J), (M), (P), and (Q)] * P ≤ 0.05, ** P < 0.01, and *** P < 0.001 by nonpaired Student’s t test. [(E) and (F)] * P ≤ 0.05 and ** P < 0.01 nonpaired Student’s t test compared with before cold stimulation.

Article Snippet: Total protein lysates (20 μg) were immunoblotted with rabbit anti–p-FAK (Y397) (1:1000; ABclonal, #AP0302), rabbit anti-FAK (1:1000; ABclonal, A11195), rabbit anti-p65 antibody (1:1000; Abcam, ab32536), rabbit anti-CCR4 antibody (1:1000; Novus Biological, NB56336SS), rabbit anti-UCP1 antibody (1:500; Abcam, ab23841), rabbit anti-tubulin antibody [1:2000; Cell Signaling Technology (CST), 2146S], mouse anti-CCL22 antibody (1:1000; R&D Systems, MAB439-SP), mouse anti-CD206 antibody (Bio-Rad, MAC2235GA), rat anti-F4/80 (1:500; Abcam, ab6640), rat anti-siglecF (1:500; Novus Biological, NBP1-91149), rabbit anti–IL-13 antibody (1:500; ABclonal, A2089), rabbit anti-p-STAT6 antibody (1:500; ABclonal, AP0456), rabbit anti-STAT6 antibody (1:500; ABclonal, A0755), rabbit anti-Histone3 antibody (1:1000; CST, 4499P), followed by goat anti-rat horseradish peroxidase (HRP)–conjugated secondary antibody (1:5000; ABconal, AS028), anti-rabbit HRP-conjugated secondary antibody (1:5000; CST, 7074S), goat anti-mouse HRP-conjugated secondary antibody (1:5000; CST, 96714S).

Techniques: Expressing, Derivative Assay, Immunofluorescence, Western Blot, Staining

( A ) Pearson’s correlation coefficient analysis of serum CCL22 concentration and fat mass ( n = 28 per group). ( B to F ) Body weight (B), energy consumption rate [(C) and (D)], and RER [(E) and (F)] ( n = 8 per group). Ten-week-old C57BL/6 male mice received saline or rCCL22 (20 μg/kg per day) at 6°C for 14 days with HFD. ( G to I ) Immunofluorescence (G), immunoblots (H) of UCP1, and mRNA expression (I) of thermogenic genes in iWAT ( n = 3 to 6 per group). Scale bar, 50 μm. ( J to L ) Immunofluorescence (J), immunoblots, and quantification [(K) and (L)] of UCP1 in iWAT ( n = 3 per group). SVF cells were extracted from HFD-fed iWAT, cultured with vehicle or rCCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes for 5 days. Scale bars, 25 μm. ( M ) Schematic diagram: The 6-month trial of obese adults participated in ADF: a 3-month weight loss period followed by a 3-month weight maintenance period. ( N and O ) Pearson’s correlation coefficient analysis of human serum CCL22 levels, body weight (N), and fat mass (O) ( n = 40). ( P ) Human serum CCL22 levels ( n = 20). ( Q ) Schematic diagram: Human SVFs from iWAT were cultured at 31°C for 5 days, treated with vehicle or CCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes. ( R to T ) Immunoblots and quantification [(R) and (S)] of UCP1 and mRNA expression (T) of thermogenic genes in human beige adipocytes ( n = 3 to 6 per group). Data information: Results are presented as means ± SEM. [(A), (N), and (O)] Two-tailed Pearson’s correlation coefficient analysis. [(F), (I), (L), (S), and (T)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. (P) ** P < 0.01 by nonpaired Student’s t test compared with month 0 body weight. (B) * P ≤ 0.05 by two-way analysis of variance (ANOVA) followed by post hoc Bonferroni tests.

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: ( A ) Pearson’s correlation coefficient analysis of serum CCL22 concentration and fat mass ( n = 28 per group). ( B to F ) Body weight (B), energy consumption rate [(C) and (D)], and RER [(E) and (F)] ( n = 8 per group). Ten-week-old C57BL/6 male mice received saline or rCCL22 (20 μg/kg per day) at 6°C for 14 days with HFD. ( G to I ) Immunofluorescence (G), immunoblots (H) of UCP1, and mRNA expression (I) of thermogenic genes in iWAT ( n = 3 to 6 per group). Scale bar, 50 μm. ( J to L ) Immunofluorescence (J), immunoblots, and quantification [(K) and (L)] of UCP1 in iWAT ( n = 3 per group). SVF cells were extracted from HFD-fed iWAT, cultured with vehicle or rCCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes for 5 days. Scale bars, 25 μm. ( M ) Schematic diagram: The 6-month trial of obese adults participated in ADF: a 3-month weight loss period followed by a 3-month weight maintenance period. ( N and O ) Pearson’s correlation coefficient analysis of human serum CCL22 levels, body weight (N), and fat mass (O) ( n = 40). ( P ) Human serum CCL22 levels ( n = 20). ( Q ) Schematic diagram: Human SVFs from iWAT were cultured at 31°C for 5 days, treated with vehicle or CCL22 (10 ng/ml) for 4 days, and then induced to beige adipocytes. ( R to T ) Immunoblots and quantification [(R) and (S)] of UCP1 and mRNA expression (T) of thermogenic genes in human beige adipocytes ( n = 3 to 6 per group). Data information: Results are presented as means ± SEM. [(A), (N), and (O)] Two-tailed Pearson’s correlation coefficient analysis. [(F), (I), (L), (S), and (T)] * P ≤ 0.05 and ** P < 0.01 by nonpaired Student’s t test. (P) ** P < 0.01 by nonpaired Student’s t test compared with month 0 body weight. (B) * P ≤ 0.05 by two-way analysis of variance (ANOVA) followed by post hoc Bonferroni tests.

Techniques: Concentration Assay, Saline, Immunofluorescence, Western Blot, Expressing, Cell Culture, Two Tailed Test

Primer sequences for qPCR gene expression analysis.

Journal: Science Advances

Article Title: Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure

doi: 10.1126/sciadv.adn5229

Figure Lengend Snippet: Primer sequences for qPCR gene expression analysis.

Techniques: Expressing

Identification of CCL22 as a target for treatment of contact allergy and development of aptamers targeting murine CCL22 (A) Time line of the CHS mouse model. (B) The ear swelling response in CCL22 −/− mice in the CHS model is significantly reduced compared to WT mice ( n = 7, mean ± SEM). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test (∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Flow cytometry-based interaction analysis of enriched DNA libraries from the SL and selection cycles 5, 8, and 10 to murine CCL22. (D) Frequency of unique sequences in selection cycles 3, 6, 8, and 10. The frequency was calculated by dividing the overall number of sequences by the number of unique sequences. (E) Number of sequence reads in the NGS analysis per selection cycle. (F) Fraction of sequences in the DNA population from selection cycles 3, 6, 8, and 10 sharing the indicated copy numbers. (G and H) Nucleotide distribution in the random region of the SL and library from selection cycle 10. The nucleotides are evenly distributed in the SL, and preferences for particular nucleotides at certain positions evolve during the selection. (I) Frequency of the most enriched sequences in selection cycles 3, 6, 8, and 10. Missing data points indicate that the sequence could not be detected in the NGS data of the respective round. (J) DNA sequences identified by NGS were analyzed for binding to murine CCL22 by ELONA ( n = 2, mean ± SD).

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Identification of CCL22 as a target for treatment of contact allergy and development of aptamers targeting murine CCL22 (A) Time line of the CHS mouse model. (B) The ear swelling response in CCL22 −/− mice in the CHS model is significantly reduced compared to WT mice ( n = 7, mean ± SEM). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test (∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Flow cytometry-based interaction analysis of enriched DNA libraries from the SL and selection cycles 5, 8, and 10 to murine CCL22. (D) Frequency of unique sequences in selection cycles 3, 6, 8, and 10. The frequency was calculated by dividing the overall number of sequences by the number of unique sequences. (E) Number of sequence reads in the NGS analysis per selection cycle. (F) Fraction of sequences in the DNA population from selection cycles 3, 6, 8, and 10 sharing the indicated copy numbers. (G and H) Nucleotide distribution in the random region of the SL and library from selection cycle 10. The nucleotides are evenly distributed in the SL, and preferences for particular nucleotides at certain positions evolve during the selection. (I) Frequency of the most enriched sequences in selection cycles 3, 6, 8, and 10. Missing data points indicate that the sequence could not be detected in the NGS data of the respective round. (J) DNA sequences identified by NGS were analyzed for binding to murine CCL22 by ELONA ( n = 2, mean ± SD).

Article Snippet: Coupling of CCL22 on carboxyl beads was validated by staining with an anti-CCL22 antibody (mouse α-murine CCL22 [R&D Systems]) and fluorescently labeled secondary antibody (goat α-mouse Alexa Fluor 647 (Jackson ImmunoResearch) by flow cytometry.

Techniques: Flow Cytometry, Selection, Sequencing, Binding Assay

Aptamer-dependent inhibition of migration toward mCCL22 in vitro and motifs enriched during CCL22 SELEX (A–H) Migration of BW5147.3 cells toward CCL22 (12.8 nM) was measured in a transwell migration assay in the presence of aptamers. Full-length aptamers AJ1 (A), AJ21 (B), AJ25 (C), AJ78 (D), AJ81 (E), AJ82 (F), AJ102 (G), and AJ104 (H) were tested in a 1:10 M ratio (1.28 nM), an equimolar ratio (12.8 nM), and a 10:1 M ratio (128 nM) along with their scrambled control sequences. As control, migration without the addition of CCL22 or aptamers, as well as the migration toward CCL22 only were measured. Statistical significance was calculated against migration toward CCL22 and tested by using ordinary 1-way ANOVA with post hoc Bonferroni test with n = 3–16 (mean ± SEM, ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (I) Frequency of 3 motifs common among the most enriched sequences. Motif 1 is found in AJ81 and AJ82. Motif 2 is found in AJ1, AJ25, and AJ104. Motif 3 is found in AJ21 and AJ102. (J) Sequences of the motifs identified by MEME Suite.

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Aptamer-dependent inhibition of migration toward mCCL22 in vitro and motifs enriched during CCL22 SELEX (A–H) Migration of BW5147.3 cells toward CCL22 (12.8 nM) was measured in a transwell migration assay in the presence of aptamers. Full-length aptamers AJ1 (A), AJ21 (B), AJ25 (C), AJ78 (D), AJ81 (E), AJ82 (F), AJ102 (G), and AJ104 (H) were tested in a 1:10 M ratio (1.28 nM), an equimolar ratio (12.8 nM), and a 10:1 M ratio (128 nM) along with their scrambled control sequences. As control, migration without the addition of CCL22 or aptamers, as well as the migration toward CCL22 only were measured. Statistical significance was calculated against migration toward CCL22 and tested by using ordinary 1-way ANOVA with post hoc Bonferroni test with n = 3–16 (mean ± SEM, ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (I) Frequency of 3 motifs common among the most enriched sequences. Motif 1 is found in AJ81 and AJ82. Motif 2 is found in AJ1, AJ25, and AJ104. Motif 3 is found in AJ21 and AJ102. (J) Sequences of the motifs identified by MEME Suite.

Techniques: Inhibition, Migration, In Vitro, Transwell Migration Assay, Control

Binding affinity and specificity of aptamer AJ82 and AJ102 (A and B) Concentration-dependent binding of aptamer AJ82 (A) and AJ102 (B) measured by SPR using murine CCL22 as analyte and biotinylated aptamers as immobilized ligands ( n = 2, mean ± SD). (C) Table of kinetic properties of the aptamers AJ82, AJ82.51, AJ102, and AJ102.29 as measured by flow cytometry or SPR. nd, not determined. (D) Interaction analysis of aptamer AJ82 and AJ102 with murine CCL22, human CCL22, murine CCL17, human CCL17, and human CCL3 measured by an ELONA ( n = 2–3, mean ± SD).

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Binding affinity and specificity of aptamer AJ82 and AJ102 (A and B) Concentration-dependent binding of aptamer AJ82 (A) and AJ102 (B) measured by SPR using murine CCL22 as analyte and biotinylated aptamers as immobilized ligands ( n = 2, mean ± SD). (C) Table of kinetic properties of the aptamers AJ82, AJ82.51, AJ102, and AJ102.29 as measured by flow cytometry or SPR. nd, not determined. (D) Interaction analysis of aptamer AJ82 and AJ102 with murine CCL22, human CCL22, murine CCL17, human CCL17, and human CCL3 measured by an ELONA ( n = 2–3, mean ± SD).

Techniques: Binding Assay, Concentration Assay, Flow Cytometry

Truncation of aptamers and specificity of AJ82.51 and AJ102.29 (A) Flow cytometry-based interaction assay of truncated variants of aptamer AJ82 with CCL22. AJ82 was truncated from 80 to 51 nt, including an initial point mutation at position 49 (dA to dC) for stabilization of the stem structure. (B and C) Structure predictions of aptamer AJ82 (B) and AJ102 (C) as predicted by Mfold web server. Shown is the prediction for the full-length sequence. Colored nucleotides indicate the motif 1 in AJ82 and motif 2 in AJ102. Gray nucleotides were truncated. (D) Flow cytometry-based interaction assay of truncated variants of aptamer AJ102 with CCL22. AJ102.29 was truncated from 80 to 29 nucleotides. AJ102.24 was truncated from the 5′ end, while AJ102.25 was truncated from the 3′ end ( n = 2, mean ± SD). (E) Flow cytometry-based interaction analysis with AJ102 and point mutants within the motif region ( n = 2, mean ± SD). Mainly dG positions are involved in target binding. (F and G) Interaction analysis of truncated 500-nM aptamers AJ82.51 (F) or AJ102.29 (G) with related chemokines human CCL22, murine and human CCL17, human CCL3, human CXCL1, human CXCL9, human CXCL10, human CXCL11 and unrelated proteins human serum albumin (HSA) and mitogen-activated protein kinase 1 (Erk2) measured by ELONA ( n = 2, mean ± SD).

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Truncation of aptamers and specificity of AJ82.51 and AJ102.29 (A) Flow cytometry-based interaction assay of truncated variants of aptamer AJ82 with CCL22. AJ82 was truncated from 80 to 51 nt, including an initial point mutation at position 49 (dA to dC) for stabilization of the stem structure. (B and C) Structure predictions of aptamer AJ82 (B) and AJ102 (C) as predicted by Mfold web server. Shown is the prediction for the full-length sequence. Colored nucleotides indicate the motif 1 in AJ82 and motif 2 in AJ102. Gray nucleotides were truncated. (D) Flow cytometry-based interaction assay of truncated variants of aptamer AJ102 with CCL22. AJ102.29 was truncated from 80 to 29 nucleotides. AJ102.24 was truncated from the 5′ end, while AJ102.25 was truncated from the 3′ end ( n = 2, mean ± SD). (E) Flow cytometry-based interaction analysis with AJ102 and point mutants within the motif region ( n = 2, mean ± SD). Mainly dG positions are involved in target binding. (F and G) Interaction analysis of truncated 500-nM aptamers AJ82.51 (F) or AJ102.29 (G) with related chemokines human CCL22, murine and human CCL17, human CCL3, human CXCL1, human CXCL9, human CXCL10, human CXCL11 and unrelated proteins human serum albumin (HSA) and mitogen-activated protein kinase 1 (Erk2) measured by ELONA ( n = 2, mean ± SD).

Techniques: Flow Cytometry, Mutagenesis, Sequencing, Binding Assay

Inhibition of CCL22 mediated cell migration in vitro by truncated and modified aptamers AJ82.51 and AJ102.29 (A) Schematic representation of the transwell migration assay. BW5147.3 cells were placed in the upper layer of the insert with a permeable membrane, and CCL22 was added at different concentrations to the lower compartment. Following an incubation period, the cells that migrated through the membrane were quantified by flow cytometry. (B and C) Migration of BW5147.3 cells toward CCL22 (12.8 nM) was measured in a transwell migration assay in the presence of aptamers. Truncated aptamers AJ82.51 (B) and AJ102.29 (C) and truncated and modified aptamers AJ82.51m (B) and AJ102.29m (C) were tested in a 1:10 M ratio (1.28 nM), an equimolar ratio (12.8 nM), and a 10:1 M ratio (128 nM). As control, migration toward the control sequences AJ82.51 ctrl, AJ102.29 ctrl and AJ82.51m ctrl, AJ102.29m ctrl (12.8 nM), without the addition of CCL22 or aptamers, as well as the migration toward CCL22 only were measured. Statistical significance was calculated against migration toward CCL22 and tested by using ordinary 1-way ANOVA with post hoc Bonferroni test, with n = 7–15 (mean ± SEM; ∗∗∗∗ p < 0.0001). ns, not significant.

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Inhibition of CCL22 mediated cell migration in vitro by truncated and modified aptamers AJ82.51 and AJ102.29 (A) Schematic representation of the transwell migration assay. BW5147.3 cells were placed in the upper layer of the insert with a permeable membrane, and CCL22 was added at different concentrations to the lower compartment. Following an incubation period, the cells that migrated through the membrane were quantified by flow cytometry. (B and C) Migration of BW5147.3 cells toward CCL22 (12.8 nM) was measured in a transwell migration assay in the presence of aptamers. Truncated aptamers AJ82.51 (B) and AJ102.29 (C) and truncated and modified aptamers AJ82.51m (B) and AJ102.29m (C) were tested in a 1:10 M ratio (1.28 nM), an equimolar ratio (12.8 nM), and a 10:1 M ratio (128 nM). As control, migration toward the control sequences AJ82.51 ctrl, AJ102.29 ctrl and AJ82.51m ctrl, AJ102.29m ctrl (12.8 nM), without the addition of CCL22 or aptamers, as well as the migration toward CCL22 only were measured. Statistical significance was calculated against migration toward CCL22 and tested by using ordinary 1-way ANOVA with post hoc Bonferroni test, with n = 7–15 (mean ± SEM; ∗∗∗∗ p < 0.0001). ns, not significant.

Techniques: Inhibition, Migration, In Vitro, Modification, Transwell Migration Assay, Membrane, Incubation, Flow Cytometry, Control

Ex vivo and in vivo application of AJ102.29 results in effective reduction of contact hypersensitivity symptoms and penetration of the skin (A) Time line of the contact hypersensitivity model application time points of AJ102.29m. (B) Ear swelling of WT mice that received PBS, 10 nmol AJ102.29m, or 10 nmol AJ102.29mctrl intraperitoneally and of CCL22 −/− mice 24 h (day 1), 48 h (day 2), and 72 h (day 3) after application of DNFB (solid lines) or vehicle (dashed lines). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test ( n = 6 WT PBS, WT AJ102.29 ctrl, WT AJ102.29 ctrl, n = 3 CCL22 −/− , mean ± SEM; ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Schematic representation of the Franz-diffusion cell assay. Fluorescently labeled aptamer in DAC cream was placed in the donor compartment. The skin of a mouse ear was placed horizontally between the donor and receptor compartment filled with RPMI medium. After 24 h the skin sample was stained with DAPI and analyzed by fluorescent micrsocopy to investigate skin penetration of the aptamer. (D) DAPI-stained sections of mouse ears treated with DAC cream with or without Atto647 labeled AJ102.29 (10 pmol mg −1 ) in an ex vivo Franz-diffusion cell assay. (E) Ear swelling of WT mice that received PBS, 10 nmol AJ102.29m, or AJ102.29mctrl mixed in a DAC cream and topically applied on the ear at the time of and 12 h after challenge, and of CCL22 −/− mice 24 h (day 1), 48 h (day 2), and 72 h (day 3) after application of DNFB (solid lines) or vehicle (dashed lines) ( n = 4–5, mean ± SEM). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test ( n = 5 per group, mean ± SEM; ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Journal: Molecular Therapy. Nucleic Acids

Article Title: Topical application of a CCL22-binding aptamer suppresses contact allergy

doi: 10.1016/j.omtn.2024.102254

Figure Lengend Snippet: Ex vivo and in vivo application of AJ102.29 results in effective reduction of contact hypersensitivity symptoms and penetration of the skin (A) Time line of the contact hypersensitivity model application time points of AJ102.29m. (B) Ear swelling of WT mice that received PBS, 10 nmol AJ102.29m, or 10 nmol AJ102.29mctrl intraperitoneally and of CCL22 −/− mice 24 h (day 1), 48 h (day 2), and 72 h (day 3) after application of DNFB (solid lines) or vehicle (dashed lines). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test ( n = 6 WT PBS, WT AJ102.29 ctrl, WT AJ102.29 ctrl, n = 3 CCL22 −/− , mean ± SEM; ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001). (C) Schematic representation of the Franz-diffusion cell assay. Fluorescently labeled aptamer in DAC cream was placed in the donor compartment. The skin of a mouse ear was placed horizontally between the donor and receptor compartment filled with RPMI medium. After 24 h the skin sample was stained with DAPI and analyzed by fluorescent micrsocopy to investigate skin penetration of the aptamer. (D) DAPI-stained sections of mouse ears treated with DAC cream with or without Atto647 labeled AJ102.29 (10 pmol mg −1 ) in an ex vivo Franz-diffusion cell assay. (E) Ear swelling of WT mice that received PBS, 10 nmol AJ102.29m, or AJ102.29mctrl mixed in a DAC cream and topically applied on the ear at the time of and 12 h after challenge, and of CCL22 −/− mice 24 h (day 1), 48 h (day 2), and 72 h (day 3) after application of DNFB (solid lines) or vehicle (dashed lines) ( n = 4–5, mean ± SEM). Data were tested for statistical significance by 2-way ANOVA with Bonferroni post hoc test ( n = 5 per group, mean ± SEM; ∗ p = 0.01–0.05; ∗∗ p = 0.001–0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Techniques: Ex Vivo, In Vivo, Diffusion-based Assay, Labeling, Cream, Staining

A . Differentially expressed genes in unstimulated or LPS or IL-4 stimulated LysM ΔZeb1 compared to LysM Ctrl BMDMs as measured by a customized RT2 array and depicted in log 2 fold change of expression. nd marks non-detectable mRNA levels. All transcripts were normalized to Gapdh (n=3). B . Relative mRNA expression of Ccl2 and Ccl22 in LysM Ctrl and LysM ΔZeb1 BMDMs (n=3; means ±SD; 2-way ANOVA). C . Comparison of transcript and secretome alterations of Ccl2 and Ccl22 in LysM ΔZeb1 compared to LysM Ctrl BMDMs (n≥5; means ±SD).

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Differentially expressed genes in unstimulated or LPS or IL-4 stimulated LysM ΔZeb1 compared to LysM Ctrl BMDMs as measured by a customized RT2 array and depicted in log 2 fold change of expression. nd marks non-detectable mRNA levels. All transcripts were normalized to Gapdh (n=3). B . Relative mRNA expression of Ccl2 and Ccl22 in LysM Ctrl and LysM ΔZeb1 BMDMs (n=3; means ±SD; 2-way ANOVA). C . Comparison of transcript and secretome alterations of Ccl2 and Ccl22 in LysM ΔZeb1 compared to LysM Ctrl BMDMs (n≥5; means ±SD).

Article Snippet: Final concentrations of cytokines were 1.93 ng/ ml CCL2 (R&D Systems, 479-JE-050) and 0.18 ng/ ml CCL22 (R&D Systems, 439-MD-025) and of antibodies 5 μg/ml IgG (Diagenode, C15410206), anti-CCL2 (Novus Biologicals, NBP1-07035SS) or anti-CCL22 (abcam, ab124768), respectively.

Techniques: Expressing, Comparison

A-B . Venn diagrams of differentially expressed genes (DEGs) of LysM Ctrl (blue) and LysM ΔZeb1 BMDMs (red) after stimulation with LPS (A) or IL-4 (B) compared to unstimulated in total (left) and divided in up-/downregulated DEGs (right). C . GO term enrichment analysis for DEGs (FDR<0.05) uniquely up- or downregulated by LysM ΔZeb1 BMDMs after LPS stimulation. D . Log 2 fold change of expression of selected trafficking genes after LPS stimulation. X marks no significant deregulation. E . Representative images and quantification of OPP incorporation of LysM Ctrl and LysM ΔZeb1 BMDMs with 0h, 4h and 16h LPS pre-stimulation (n=3; means ±SD; 2-way ANOVA). F-G . Representative arrays of intracellular cytokines of LysM Ctrl and LysM ΔZeb1 BMDMs with LPS stimulation or additional Brefeldin A and Monensin treatment (F) and quantification of intracellular CCL2 and CCL22 after LPS, Brefeldin A and Monensin treatment (G; n≥2).

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A-B . Venn diagrams of differentially expressed genes (DEGs) of LysM Ctrl (blue) and LysM ΔZeb1 BMDMs (red) after stimulation with LPS (A) or IL-4 (B) compared to unstimulated in total (left) and divided in up-/downregulated DEGs (right). C . GO term enrichment analysis for DEGs (FDR<0.05) uniquely up- or downregulated by LysM ΔZeb1 BMDMs after LPS stimulation. D . Log 2 fold change of expression of selected trafficking genes after LPS stimulation. X marks no significant deregulation. E . Representative images and quantification of OPP incorporation of LysM Ctrl and LysM ΔZeb1 BMDMs with 0h, 4h and 16h LPS pre-stimulation (n=3; means ±SD; 2-way ANOVA). F-G . Representative arrays of intracellular cytokines of LysM Ctrl and LysM ΔZeb1 BMDMs with LPS stimulation or additional Brefeldin A and Monensin treatment (F) and quantification of intracellular CCL2 and CCL22 after LPS, Brefeldin A and Monensin treatment (G; n≥2).

Techniques: Expressing

A . Confluence of KPC cells alone or co-cultured with LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). B . Representative images at t=28h and quantification over time of KPC cell invasion into a scratch wound without or with co-culture of LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). C . Confluence of KPC cells alone or with LysM Ctrl or LysM ΔZeb1 BMDM conditioned medium (CM) (n=2 KPC CM, n=3 LysM Ctrl and LysM ΔZeb1 CM). D . Transwell migration assay of CD8+ T cells alone or towards LysM Ctrl or LysM ΔZeb1 BMDMs in absence or presence of recombinant CCL2 and CCL22 (left panel, n>3) or absence or presence of anti-CCL2 and anti-CCL22 antibodies (right panel, n=3). Means ±SD; *:p<0.05; **:p<0.01; ns: not significant; 2-way ANOVA.

Journal: bioRxiv

Article Title: Macrophages foster adaptive anti-tumor immunity by ZEB1-dependent cytotoxic T cell chemoattraction

doi: 10.1101/2024.02.26.582102

Figure Lengend Snippet: A . Confluence of KPC cells alone or co-cultured with LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). B . Representative images at t=28h and quantification over time of KPC cell invasion into a scratch wound without or with co-culture of LysM Ctrl or LysM ΔZeb1 BMDMs (n=3). C . Confluence of KPC cells alone or with LysM Ctrl or LysM ΔZeb1 BMDM conditioned medium (CM) (n=2 KPC CM, n=3 LysM Ctrl and LysM ΔZeb1 CM). D . Transwell migration assay of CD8+ T cells alone or towards LysM Ctrl or LysM ΔZeb1 BMDMs in absence or presence of recombinant CCL2 and CCL22 (left panel, n>3) or absence or presence of anti-CCL2 and anti-CCL22 antibodies (right panel, n=3). Means ±SD; *:p<0.05; **:p<0.01; ns: not significant; 2-way ANOVA.

Techniques: Cell Culture, Co-Culture Assay, Transwell Migration Assay, Recombinant

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