Nesse et al. Acta Veterinaria Scandinavica 2011, 53:44 http://www.actavetscand.com/content/53/1/44 may influence the regulation of virulence traits in Sal- monella like cell invasion. In the present work, the effect of different AHLs on invasion of HEp-2 epithelial cells by Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) was studied in vitro. Materials and methods Bacterial strains The bacteria used in this study were the wildtype S. Typhimurium ATCC 14028 and its isogenic sdiA mutant (ΔSTM1950: Kan-PT7) which was kindly pro- vided by Professor McClelland at Vaccine Research Institute of San Diego, USA. As a control bacterium, known to be non-invasive, Escherichia coli ATCC 25922 was included. Cultures were routinely grown in 5 mL Luria-Bertani (LB) (Merck KGaA, Darmstadt, Germany) broth without agitation for approximately 18-20 hours at 37°C before used in experiments. Cell lines, culture conditions and buffers The cell line HEp-2 ATCC CCL-23 (LGC Standards, Middlesex, UK) was used in the invasion experiments. The cells were grown in Minimum Essential Medium (MEM; Lonza, Basel, Switzerland) supplemented 2 mM L-glutamine (Lonza) and 10% fetal calf serum (Merck) at 37°C under standard tissue culture conditions, with- out CO2 in 25 cm2 flasks (Corning B.V. Lifesciences, Amsterdam, Netherlands) and confluent flasks were split twice a week by trypsin-EDTA (Lonza) treatment and diluted 1:8 in fresh media. When used for bacterial invasion studies, the cells were diluted 1:2, seeded in 24 well plates (Corning) and incubated over night at 37°C. The following day, cells were counted using a Bürker chamber after staining with Trypan Blue Stain 0.4% (Lonza) to quantify the mean number of cells per well. Cell invasion studies Over night cultures were diluted 1:100 in 5 mL LB and subcultured for 3 hours in 37°C. Then the cultures were ten fold diluted twice, first in MEM and subsequently in MEM supplemented with either N-hexanoyl-DL-homo- serine lactone (C6-AHL) (Sigma-Aldrich, St. Louis, MO, USA) or N-octanoyl-DL-homoserine lactone (C8-AHL) (Sigma-Aldrich) to a final concentration of 1 μM/mL (hereafter called MEMs). Distilled water (dH2O) was used instead of AHL for the controls. To measure bac- terial invasion, a method based on the one described by Lissner et al. was used [6]. Briefly: 1 mL bacteria/MEMs was added to HEp-2 cells grown over night in 24 well plates, to a multiplicity of infection (MOI) of approxi- mately 100 bacteria pr cell. Plates were incubated for 90 min at 37°C and MEMs was removed. The cells were washed three times with 1 mL PBS de Boer (Na2HPO4 × 2H2O 1.34 g/L, NaH2PO4 × H2O 0.34 g/L, NaCl 8.5 Page 2 of 5 g/L, pH: 7.2 ± 0.1). Then 1 mL MEM supplemented with 250 μg/mL gentamycin (Lonza) (hereafter called MEMg) was added, followed by incubation at 37°C for two hours. MEMg was removed; cells were washed three times with PBS de Boer and lysed with ice-cold 1% Triton-X (Sigma-Aldrich) in PBS de Boer for 10 min. The cells were dislodged using a sterile cell scraper (BD Falcon, Bedford, MA, USA), pipetted to disperse bacterial aggregates, ten fold serial diluted and plated out on blood agar to determine the number of cfu. In each experiment, every combination of supplement and bacteria were tested in triplets, and three indepen- dent experiments were performed for each strain. To eliminate the day to day variations, the effect of AHL addition in each experiment was calculated as fold change, i.e.: (the mean number of intracellular cfu after addition of AHL)/(the mean number of intracellular cfu without addition of AHL), and thereafter log10 trans- formed to allow calculation of confidence intervals. Results are given as means of three independent experi- ments. An increase in cell invasion was considered sta- tistically significant (p <0.05) if the value “0” was not included in the 95% confidence interval. Semi-quantitative PCR The bacteria were prepared and incubated with 1 μM AHL or dH2O as described under cell invasion studies with the exceptions that HEp-2 cells were not present, and the incubation was performed in 100 mL LB. After incubation, each bacterial suspension was divided into 25 mL aliquots and transferred to four 50 mL test tubes (Greiner Bio-One, Frickenhausen, Germany). To each tube, 5 mL ice cold 5% acidic phenol (Sigma Aldrich)/ 95% ethanol (Kemetyl Norge, Vestby, Norway) were added and the mixture was kept on ice for 20 min to stabilize the mRNA [7]. The mixture was then centri- fuged at 4°C, 2330 × G for 20 min, and most of the supernatant discarded. Each pellet was resuspended in the remaining supernatant. The suspensions were pooled two and two, transferred to two Eppendorf tubes (BRAND GMBH, Wertheim, Germany) and centrifuged at 14 000 × G for 1 min at room temperature. The supernatants were discarded and the pellets frozen at -70°C until the next step in the procedure. Total RNA was isolated from the pellets using a SV Total RNA Iso- lation kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s instructions. cDNA was synthesized immediately after RNA isolation, using Invi- trogen’s SuperScript® II Reverse Transcriptase (Invitro- gen, Ltd, Paisley, UK) according to manufacturer’s instructions. PCR was performed in 25 μL reaction volumes using1 μL cDNA, 0.5 μL of each primer, 0.5 μL of each dNTP and 0.2 μL Taq polymerase (Qiagen GmbH, Hilden, Germany), and thermocycled by 5 min