enzymatic assay  (Biothema AB)

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