168 Chapter 5 using Bio-Plex Manager Software (Bio-Rad Laboratories, Veenendaal, The Netherlands). Values below the minimum of the standard were based on extrapolation of the standard curve by the software. Statistical analyses Distribution of the data was checked for normality using the Shapiro Wilk test. For the FCM and IHC data, differences between burn tissue and healthy skin, and between burn tissues of different time intervals after injury (PBW 1, 2, 3 and 4) were explored using the Mann Whitney U test in Graphpad version 5.01 (PRISM, La Jolla, USA). Only statistically significant differences are shown and are indicated by black asterisks (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). The data was visualized using Graphpad version 5.01 (PRISM, La Jolla, USA). Levels of the soluble immune factors in burn tissue were transformed to fold change differences compared to the levels in healthy skin. P values of differences between burn tissue and healthy skin were determined using Mann Whitney U test. We considered a p value of < 0.05 to be statistically significant. Volcano plots were created using “EnhancedVolcano” version 1.6.0 package in R version 3.6.2. ACKNOWLEDGMENTS We would like to thank Xuehui He for helping with the cell isolation protocol, Bram van Cranenbroek for his assistance on the immunoassay, Mark Gorris for helping with the multiplex imaging and Evi Warmerdam, Rosa Rentenaar and Myrthe Witbaard for their technical assistance during experiments. We are grateful for the work and participation of all involved physicians, surgeons, nurses and patients of the Burn Center and the Department of Plastic and Reconstructive Surgery of the Red Cross Hospital. This research was funded by the Dutch Burns Foundation under grant number WO/17.108 (BKHLB).
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