Bacterial Culture and Inflammatory Modulator Screening

Cultures of E. coli (isolate MS499)^{16 (link)} or T. pyogenes (isolate MS249),^{17 (link)} previously collected from the uteri of postpartum cows with persistent uterine disease, were grown overnight in Luria-Bertani medium (Sigma-Aldrich) and brain-heart infusion medium (Sigma-Aldrich) supplemented with 5% FBS, respectively, as described previously.^{6 (link), 18 (link)} The concentration of bacteria was measured by colony count and suspended to 1 × 10⁸ colony forming units (CFU)/ml in sterile PBS (Life Technologies Ltd, Paisley, UK), followed by centrifugation at 6,000 × g for 10 min at 4°C. After washing, bacteria were diluted to 1 × 10³ CFU/ml (E. coli) or 1 × 10⁸ CFU/ml (T. pyogenes) in complete medium. Ultrapure LPS from E. coli 0111:B4 was obtained from InvivoGen (Toulouse, France), and used at 100 ng/ml, as this concentration has previously been shown to be optimal for stimulating IL-6 and CXCL8 responses in endometrial cells.^{19 (link)} Full details of the small molecules used as a part of the inflammatory modulator screening are provided in Supplemental Table I. The isoprenoid alcohols farnesol and geranylgeraniol were obtained from Sigma-Aldrich.