Journal: Scientific reports

Article Title: Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation.

doi: 10.1038/s41598-024-81014-9

Figure Lengend Snippet: Fig. 4. Integration of Mutz-LCs into a full-thickness skin model. (A) H&E staining of the full-thickness skin model including LC surrogates. Scale Bar = 100 µm. (B): Immunofluorescent staining skin model including LC surrogates. LC surrogates were stained with CD1a (yellow signal) and nuclei were stained with DAPI (Blue signal). Scale bar = 20 µm. (C-D) Analysis of the relative mRNA levels (ΔCq) of LC markers, maturation and migration markers and cytokines expressed by the epidermal (C) and dermal (D) compartment in the regular full-thickness skin model vs. the full-thickness skin model with incorporated LC surrogates. Epidermis and dermis of the full-thickness model without and with incorporated LC surrogates were separated and dissociated enzymatically and RNA was extracted for cDNA synthesis for RT-qPCR. Error bars indicate the standard errors of the mean (n=3 independent experiments with *p ≤ 0.05, ***p ≤ 0.001 and ****p ≤ 0.0001)

Article Snippet: At least 1 × 105 cells for each antibody panel were transferred to 96-well u-bottom plates and incubated in Automacs Running Buffer with the following antibodies (1:50): REA Control (S)-VioGreen (Miltenyi Biotec, #130-113-444), REA Control (S)-PE (Miltenyi Biotec, #130-113-438), REA Control (S)-APC (Miltenyi Biotec, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi Biotec, #130- 113-440); HLA-DR-VioGreen (Miltenyi Biotec, #130-111-948), CD1a-PE (Miltenyi Biotec, #130-112-022); CD207-PE-Vio770 (Miltenyi Biotec, #130-112-370), CD54-APC (Miltenyi Biotec, #130-121-342); CD86-APC (Miltenyi Biotec, #130-116-161), CD83-PE (Miltenyi Biotec, #130-110-561), CD11b-VioGreen (Miltenyi Biotec, #130-110-617), CD11c-APC (Miltenyi Biotec, #130-113-584) for 10 min in the dark.

Techniques: Staining, Migration, cDNA Synthesis, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation.

doi: 10.1038/s41598-024-81014-9

Figure Lengend Snippet: Fig. 5. Histological analysis of the full thickness skin model with incorporated LC surrogates. Skin models were topically treated with NiSO4 [380 µM] and DNCB [20 µM] for 24 h. (A) Immunofluorescent staining of the full-thickness skin model tissue including LC surrogates after treatment with solvent control or sensitizers. LC surrogates were stained with CD1a (yellow signal). Nuclei were stained with DAPI (blue signal). Scale bar = 20 µm. Sensitizer induced migration of the LC surrogates from the epidermis to the dermal compartment was quantified via whole slide image analysis and depicted as fold of induction for CD1a positive cells in the epidermis and dermis compared to the solvent control (B) and as distribution in percentage (C). Error bard indicate the standard errors of the mean (n=3 independent experiments with *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001)

Article Snippet: At least 1 × 105 cells for each antibody panel were transferred to 96-well u-bottom plates and incubated in Automacs Running Buffer with the following antibodies (1:50): REA Control (S)-VioGreen (Miltenyi Biotec, #130-113-444), REA Control (S)-PE (Miltenyi Biotec, #130-113-438), REA Control (S)-APC (Miltenyi Biotec, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi Biotec, #130- 113-440); HLA-DR-VioGreen (Miltenyi Biotec, #130-111-948), CD1a-PE (Miltenyi Biotec, #130-112-022); CD207-PE-Vio770 (Miltenyi Biotec, #130-112-370), CD54-APC (Miltenyi Biotec, #130-121-342); CD86-APC (Miltenyi Biotec, #130-116-161), CD83-PE (Miltenyi Biotec, #130-110-561), CD11b-VioGreen (Miltenyi Biotec, #130-110-617), CD11c-APC (Miltenyi Biotec, #130-113-584) for 10 min in the dark.

Techniques: Staining, Solvent, Control, Migration

Journal: Scientific reports

Article Title: Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation.

doi: 10.1038/s41598-024-81014-9

Figure Lengend Snippet: Fig. 7. Histological analysis of the full-thickness skin model with incorporated LC surrogates and DDC surrogates. (A) Immunofluorescent staining of the immune competent full-thickness skin model including LC and DDC surrogates. LC surrogates were stained with CD1a (yellow signal). DDC surrogates were stained with CD45 (red signal) and Nuclei were stained with DAPI (Blue signal). Scale bar = 20 µm. (B) Quantification of the CD1a signal after topical treatment with NiSO4 and DNCB for 0h, 8 h, and 24 h was achieved via whole slide image analysis and depicted as fold of induction of CD1a positive cells for the epidermal compartment and the dermal compartment. Error bars indicate the standard errors of the mean (n = 3) independent experiments with each two technical replicates and with *p ≤ 0.05 and **p ≤ 0.01).

Article Snippet: At least 1 × 105 cells for each antibody panel were transferred to 96-well u-bottom plates and incubated in Automacs Running Buffer with the following antibodies (1:50): REA Control (S)-VioGreen (Miltenyi Biotec, #130-113-444), REA Control (S)-PE (Miltenyi Biotec, #130-113-438), REA Control (S)-APC (Miltenyi Biotec, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi Biotec, #130- 113-440); HLA-DR-VioGreen (Miltenyi Biotec, #130-111-948), CD1a-PE (Miltenyi Biotec, #130-112-022); CD207-PE-Vio770 (Miltenyi Biotec, #130-112-370), CD54-APC (Miltenyi Biotec, #130-121-342); CD86-APC (Miltenyi Biotec, #130-116-161), CD83-PE (Miltenyi Biotec, #130-110-561), CD11b-VioGreen (Miltenyi Biotec, #130-110-617), CD11c-APC (Miltenyi Biotec, #130-113-584) for 10 min in the dark.

Techniques: Staining

Journal: Nature Communications

Article Title: Impaired expression of metallothioneins contributes to allergen-induced inflammation in patients with atopic dermatitis

doi: 10.1038/s41467-023-38588-1

Figure Lengend Snippet: A – F Frequency of immune cells in control and HDM patch tests from reactive vs. non-reactive patients, measured by flow cytometry. The number in the graph indicates the percentage of cells in the positive gate. CR control patch, reactive patient, HR HDM patch, reactive patient, CNR control patch, non-reactive patient, HNR HDM patch, non-reactive patient. Representative examples. A , B CD3+ T lymphocytes, D , E CD207/CD1a positive LCs. C , F Fold changes (FC) in the percentage of detected immune cells between HDM patch test and control patch test from patients with irritant, non-reactive and reactive reactions to HDM. G Correlations between fold changes in the percentage of CD3+ T cells and LCs. Pearson correlation coefficient is shown. H Immunofluorescence staining of HDM-reactive patch test site. Inserts show the indicated optical fields at the epidermis (top) and in the dermis (bottom). Hub structures of co-localising CD207 (green) and CD3 (red) in the dermis. Epidermal layer stained with multi-cytokeratin (blue). DAPI stain for nuclei (grey). Scale bars: 500 μm, 50 μm (insets). A representative of n = 3 individual donors. I Functional assessment of skin barrier: TEWL measurements across patient groups. J Number of irritant (IR), non-reactive (NR) and reactive (R) cases with loss of function (LoF) variants in FLG compared to wildtype (WT). K Percentage of CD3+ T cells in control patch test sites identified by flow cytometry. Statistical significance was assessed by t -test. C , G NR n = 11, R n = 10, F , K NR n = 11, R n = 11, I , J IRR n = 4, NR n = 12, R n = 11. Statistical significance was assessed by the Kruskal–Wallis test with post hoc Dunn test ( C , F , I ) and unpaired ANOVA with post hoc Fisher test ( K ) following the normality Kolmogorov–Smirnov test of data distribution. Source data are provided as a Source Data file.

Article Snippet: FACS Aria flow cytometer (Becton Dickinson, USA) was used for the analysis of human LCs for the expression of CD207, CD1a, HLA-DR (mouse monoclonal antibodies, CD1a, CD207:Miltenyi Biotech, UK and HLA-DR: BD Biosciences, UK) or T cells for the expression of CD3, CD25 and CD103 (Miltenyi Biotech).

Techniques: Control, Flow Cytometry, Immunofluorescence, Staining, Functional Assay

Journal: Mediators of Inflammation

Article Title: The Influence of Ouabain on Human Dendritic Cells Maturation

doi: 10.1155/2014/494956

Figure Lengend Snippet: CD83 and HLA-DR expression on dendritic cells during activation. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF- α (50 ng/mL), in the presence or absence of 20 μ M of p38 inhibitor (SB202109). CD83 and HLA-DR expression were analyzed by flow cytometry. Data are expressed as (a) the percentage of CD83 + cells, or (b) MFI (mean of fluorescence intensity) of CD83 + cells, (c) MFI of HLA-DR + cells, and lines denote the means of 3–5 independent experiments. ∗ is significantly different from TNF- α ( ∗ P < 0.05).

Article Snippet: Cells were stained for 30 min at 4°C with FITC conjugated anti-CD14, FITC conjugated anti-HLA-DR, PE conjugated anti-CD1a, PE conjugated anti-CD86, PE conjugated anti-CD80, and PE conjugated anti-CD83 (all from BD Biosciences, USA).

Techniques: Expressing, Activation Assay, Incubation, Flow Cytometry, Fluorescence

Journal: Mediators of Inflammation

Article Title: The Influence of Ouabain on Human Dendritic Cells Maturation

doi: 10.1155/2014/494956

Figure Lengend Snippet: HLA-DR expression on dendritic cells during activation. After the differentiation period (5 days), cells were incubated for further 48 h with or without TNF- α (50 ng/mL), in the presence or absence of 100 nM Oua. HLA-DR expression was analyzed by flow cytometry. Data are expressed as (a) the percentage of HLA + cells, or (b) MFI (mean of fluorescence intensity), and lines denote the means of seven independent experiments. ∗ and ∗∗ are significantly different from the control ( ∗ P < 0.05; ∗∗ P < 0.01).

Article Snippet: Cells were stained for 30 min at 4°C with FITC conjugated anti-CD14, FITC conjugated anti-HLA-DR, PE conjugated anti-CD1a, PE conjugated anti-CD86, PE conjugated anti-CD80, and PE conjugated anti-CD83 (all from BD Biosciences, USA).

Techniques: Expressing, Activation Assay, Incubation, Flow Cytometry, Fluorescence