To assess this possibility, macrophage viability during the time frame set was assessed
by determining ATP levels (Crouch et al., 1993; Dexter et al., 2003) of stimulated cells (6- and 24-h postinfection/stimulation), using a 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazoliumbromide spectrophotometric assay (Mossmann, 1983). ATP levels of stimulated cells were compared with those on nonsimulated control cells as determined with the (bioluminescent) Vialight Plus assay (Cambrex Bio Science, Rockland, ME). For TNF-α, which possesses two biologically active isoforms (TNF-α1 and TNF-α2; Zou et al., 2002) whose functional roles are poorly understood (Bridle et al., 2006), two sets of primers designed by Purcell et al. (2004) were used. IL-1β primers are also from the same source. IL-6 primers [IL6AZ-1 (TTTGCTCCGCCTCCAACAAG) and IL6AZ-2 (GGTCTTTGACCAGCCCTATCAG)] were designed using the primer express software v2.0 (Applied Biosystems) from buy Napabucasin sequences deposited in GenBank (accession number DQ866150). Relative cytokine mRNA levels were determined by normalization of the signal with that for β-actin (Zou et al., 2004). The suitability of β-actin gene as a normalizing gene was compared with that of several other known housekeeping genes, namely: this website elongation factor-1α (EF-1α), rainbow trout histone
H2A and rainbow trout 18S rRNA gene (see Real-time PCR data analysis). As a detection system that quantitates fish cytokines is not available, and as most cytokines are transcriptionally regulated (Brorson et al., 1991), cytokine induction and quantification were assessed through cytokine mRNA transcript
levels (Livak & Schmittgen, 2001). Unrelated studies, comparing the rise of mRNA transcripts with the (ELISA) quantification of cytokines have shown that these correlate (Cui et al., 2000), and that the assay is reproducible (Stordeur et al., 2002; O’Dwyer et al., 2006). Total RNA was extracted from isolated RTS-11 cells and pronephros using the peqGOLD TriLFast™ (Peqlab), following the manufacturer’s instructions. RNA was then eluted in 200 μL of RNAse-free water, quantified MycoClean Mycoplasma Removal Kit by Nanodrop (ND 1000) and stored at −80 °C until use. The synthesis of cDNA was initiated by incubating 500 ng of RNA with 5 mM dithiothreitol (ABgene), 1 U of RNasin (Promega) and 0.25 U of RNAse-free Cloned DNAse I (Takara) for 30 min at 37 °C followed by 10 min at 65 °C. Next, 500 ng of oligo (dT) primer and 400 ng of random primers (ABgene) were added and annealed at 70 °C for 5 min, and for 5 min on ice. Finally, 5 mM dNTPs (ABgene), reverse transcriptase buffer and 50 U of Reverse-iT™ RTase blend (ABgene) were added, and the mixture was incubated for 50 min at 47 °C; the mixture was then incubated at 75 °C for 10 additional minutes. To minimize variation, all samples representing a single time point were run from the same bulk cocktail of cDNA synthesis reagents.