Fastprep 24 and lysing matrix d

Mesenteric lymph nodes (MLN) were snap‐frozen in liquid nitrogen. Samples were homogenized using a FastPrep 24 and lysing matrix D (MP Biomedicals, Illkirch, France) and total RNA extracted using RNABee (AmsBio, Abingdon, UK). The total RNA concentration was measured by absorbance at 260 nm on a NanoDrop ND‐1000 spectrophotometer (Labtech International, East Sussex, UK). Samples were treated with RNase‐free DNase (Promega, Southampton, UK) to remove any contaminating genomic DNA. Total RNA (1.0 μg) was then reverse‐transcribed using SuperScript® III First‐Strand Synthesis System for RT‐PCR (Life Technologies, Waltham, MA, USA) in a final volume of 50 μL according to the manufacturer's instructions. qRT‐PCR was performed using FastStart Universal SYBR Green Master (Rox) (Sigma‐Aldrich, Poole, Dorset, UK) and the primers listed in Table 1 on an MX3005P qPCR machine (Agilent Technologies LDA UK Ltd, Stockport, Cheshire, UK) with MxPro software (Agilent Genomics). Expression levels were determined relative to Rpl19 expression using the ΔΔCt method. Gene expression data were then normalized so that the mean expression level of each gene of interest in uninfected CXCR5^F/F control mice was 1.0.