Marilen Benner

GESTATIONAL ANTIBIOTICS AFFECT IMMUNITY 197 7 used. Pregnant mice were killed in a separate part of the flowhood to avoid contamination of the location where murine tissues were collected. The skins of the mice were carefully swabbed with ethanol, after which an intraperitoneal lavage was carried out by flushing the peritoneal cavity with 2 ml of PBS to collect intraperitoneal leukocytes. Hereafter, the abdominal cavity was opened using one set of surgical instruments, carefully avoiding contact of the skin with the abdominal cavity by pinning the skin of the animals back. Another set of sterilized surgical instruments was used to isolate placental and fetal tissues, and amniotic fluid was collected from individual amniotic cavities of the fetuses. Hereafter, other tissues (e.g. spleen, intestinal tissue and lymph nodes) were isolated, for which purpose the carcass of the mouse was moved to another section of the laboratory, to avoid contamination of the laminar flowhood. Samples isolated for analysis of mRNA-expression were immediately snap-frozen using dry ice and stored at -80°C. Samples used for flow cytometry or cell culturing were kept on ice until further processing. Microbiota-analysis placenta & cecum Total DNA was isolated from 50-225 microgram of cecal content feces and 125-200 microgram of placental tissue using the QIAamp Stool DNA mini kit (Qiagen). DNA was quantified by NanoDrop assay. The 16S rRNA gene profiling was analyzed as described by Paganelli et al 2019, by the Exposome HUB Utrecht. Briefly, 16S rRNA regions V3 and V4 were sequenced with an Illumina MiSeq reagent Kit v3 (600 ‐ cycle) on an Illumina MiSeq instrument (Illumina)(67). Samples were analyzed with the QIIME™ 2 microbial community analysis pipeline (68). For the cecum samples, significant differences between treatment and control groups at genus level were detected using the statistical framework analysis of composition of microbiomes (ANCOM)(69). p ‐ values were adjusted for multiple comparisons using false discovery rates. RStudio 1.4.1103 (RStudio Team) was used to calculate alpha diversity using the Shannon index, and significance was calculated by the Wilcoxon test. The global difference in microbiota composition was assessed using principal component analysis (PCA), employing zCompositions, centered log ‐ ratio (CLR) transformation, and ggplot R packages. SCFA analysis cecal content The cecal SCFA levels of acetic, propionic, butyric, isobutyric and valeric acids were quantitatively determined as well as levels of lactic acids as described previously (70, 71). The SCFA were captured using a Shimadzu GC2010 gas chromatograph (Shimadzu Corporation, Kyoto, Japan) equipped with a flame ionisation detector. SCFA concentrations were determined using 2-ethylbutyric acid as an internal standard. Lactic acids were determined enzymatically using a d/l-lactic acid detection kit with d- and l-lactate dehydrogenase (EnzyPlus, BioControl Systems, Inc., Bellevue, WA, USA).