225 Immune Cell Model for Burn Immune Reactions at room temperature for 30 min. After washing, detection was established using 3,3′-diaminobenzidine (DAB). After DAB staining was completed, sections were counterstained with hematoxylin, dehydrated and mounted with Eukit Mounting Medium (Sigma-Aldrich). Microscopy Microscopic visualization was performed with a Zeiss Axioskop40FL microscope (Zeiss, Breda, The Netherlands). Images were acquired using a Nikon Eclipse TS2 camera and the NIS-Elements software version 4.4 (Nikon Instruments, Amsterdam, The Netherlands). Immunoassay Cytokines, chemokines and growth factors were analyzed in samples of medium. Neat samples were measured using the Human Essential Immune Response LegendPlex Multi-analyte Flow Assay kit (cat. 740929, BioLegend), according to the manufacturer’s instructions and were acquired on the flow cytometer. This 13-plex immunoassay included: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IP-10 (CXCL10), MCP-1 (CCL2), TNF-α and TGF-β1. Concentrations were determined using FlowLogic software. When cytokine levels were lower than the standard range, the lowest level of quantification was used. When cytokine levels were higher than the standard range, the levels were estimated based on the fluorescent signal in the assay. Statistical analysis and data visualization Differences in cell number/percentages and cytokines levels between different modeling conditions were explored using Wilcoxon signed-rank test in R (ggpubr and ggplot2 packages, open source). Data was visualized using R (ggplot2 package, open source) and significant (p value of < 0.05) differences were indicated by asterisks. ACKNOWLEDGMENTS MatriDerm® was kindly provided as research material by MedSkin Solutions, Dr. Suwelack AG, Billerbeck, Germany. This research was funded by the Dutch Burns Foundation under grant numbers WO/17.108 (BKHLB) and WO/22.106 (PPGM). 7
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