For all experimental assays, 24-well tissue culture plates (Greiner) were seeded with 5.0 × 104 HEp-2 cells. Plates were incubated for 18 h at 37 °C in a humidified 5% CO2 incubator. Before the assays, the semi-confluent monolayers were washed and incubated with RPMI Medium 1640 containing 1% fetal bovine serum. Planktonic and biofilm SS cells were suspended in fresh medium to a final concentration of 107 cells mL1. Bacterial CFU were confirmed by plating onto THB agar. Cells were aliquoted
into 24-well tissue culture plates (1 mL per well). All 24-well plates were incubated under a 5% CO2 atmosphere at 37 °C for 3 h to allow cells to attach. After 3 h of incubation, all plates were washed three times with PBS to remove any nonadhering bacteria. Adherent cells were detached using 0.25% trypsin, serially diluted 10-fold in sterile PBS and plated onto THB agar plates. Results are expressed as the selleck chemicals average number of bacteria adhering to HEp-2 cells for determinations. Negative control wells containing only HEp-2 cells were used in all experiments. The assay was performed at least three times. Planktonic cultures were grown in THB medium with 1% fibrinogen at 37 °C MK-2206 order and were collected after 24 h culture time. For biofilm cultures, SS2 HA9801 was grown
in a 100-mm tissue culture dish (Greiner) at 37 °C for 24 h. Planktonic and biofilm cells were harvested and total cellular RNAs were extracted as described previously with little modification (Shemesh et al., 2007). Total RNA was extracted with an E.Z.N.A.™ Bacterial RNA isolating kit (Omega, Beijing, China) according to the manufacturer’s protocol.
The RNA was subjected to DNase I (Promega, Madison, WI) treatment to exclude genomic DNA contaminants. cDNA synthesis was performed using the PrimeScript™ RT reagent kit (TaKaRa) according to the manufacturer’s instructions. mRNA levels were measured using two-step relative qRT-PCR. Relative copy numbers and expression ratios of selected genes were normalized to the ID-8 expression of one housekeeping gene (16S rRNA gene) and calculated as described by Gavrilin et al. (2000). The housekeeping gene (16S rRNA gene) was used as an internal control for specific primers. The specific primers used for the various RT-PCR assays are listed in Table 1. The SYBR Green PCR method was used according to the SYBR Premix Ex Taq™ Kit (TaKaRa). Reactions were carried out in triplicate. An ABI 7300 RT-PCR system was used for relative qRT-PCR. Planktonic and biofilm cells were inactivated for 1 h at 90 °C (Azad et al., 1999). Efficiency of inactivation was determined by plating the above bacterial suspension onto THB and the presence of bacterial colonies was monitored for 3 days. Adult zebrafish were divided randomly into three groups (100 fish per group).