microbeads  (Miltenyi Biotec)

Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Representative histograms from flow cytometry showing binding of anti-C-C motif chemokine receptor 8 (CCR8)-positive IgG from systemic sclerosis (SSc) patients or healthy controls (HC) to CCR8-overexpressing HEK293 cells. A fluorophore-conjugated anti-human IgG Fc antibody was used for detection. B Quantification of mean fluorescence intensity (MFI) in CCR8-overexpressing cells incubated with anti-CCR8-positive or control IgG, with or without preincubation with blocking anti-CCR8 monoclonal antibody ( n = 4 technical replicates per group). C ERK phosphorylation levels in CCR8-overexpressing HEK293 cells after stimulation with CCL1 in the presence of anti-CCR8-positive or control IgG, as measured by ELISA ( n = 8 biological replicates per group). D Treg migration assay in Transwell culture systems. The number of migrated Tregs in response to CCL1 with anti-CCR8-positive IgG or control IgG ( n = 6 biological replicates per group) are presented. Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Flow Cytometry, Binding Assay, Fluorescence, Incubation, Control, Blocking Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Migration, Comparison

Journal: Nature Communications

Article Title: Autoantibody landscape and functional role of anti-C-C motif chemokine receptor 8 autoantibodies in systemic sclerosis: post-hoc analysis of a B-cell depletion trial

doi: 10.1038/s41467-025-66974-4

Figure Lengend Snippet: A Experimental protocol of the skin sclerosis mouse model. Six-week-old female C57BL/6NcrSlc mice (MGI ID: MGI:5295404) received daily subcutaneous injections of bleomycin (BLM, 200 µg) or PBS as a control for two weeks. Mice were also treated intraperitoneally with either control (Ctrl) IgG or anti-C-C motif chemokine receptor 8 (CCR8) antibody (Ab) once on Day 8. Skin biopsies were collected at Week 2 for histological analysis and next-generation sequencing (NGS). This figure was partially created with BioRender ( https://BioRender.com/slxff50 ). Representative histological images and quantitative analyses of dermal fibrosis and Treg infiltration in mouse skin. H&E-stained B and Masson’s trichrome–stained C skin sections show increased dermal thickness and collagen deposition following BLM treatment. Images were captured at ×200 magnification, and scale bars represent 50 μm. D Quantification of dermal thickness and E Foxp3⁺ Treg counts are presented in the accompanying bar graphs ( n = 6 biological replicates for PBS group; n = 5 biological replicates for BLM-treated groups). Statistical significance was assessed using Welch’s ANOVA with Dunnett’s T3 multiple comparison adjustment. Error bars are defined as the standard error of the mean. Source data are provided as a Source Data file.

Article Snippet: Tregs were isolated from human peripheral blood using the MACSxpress Whole Blood Treg Isolation Kit (#130-109-557, Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany), following the manufacturer’s instructions with minor modifications.

Techniques: Control, Next-Generation Sequencing, Staining, Comparison