Cell culture grade

All reagents were purchased from Thermo-Fisher Scientific except where otherwise noted. All DMSO utilized was cell culture grade (Sigma). RT-PCR data was generated using a CFX Connect Real-Time PCR Detection System (Biorad, Hercules, CA). For assays requiring absorbance and fluorescence measurements, a SpectraMax M2 plate reader was used (Molecular Devices, San Jose, CA).

To test the effects of eATP on cytokine and immune-related gene expression,
Japanese flounder HKM and PBL cells were treated with 200 or 1000 μM ATP (cell
culture grade, Sigma-Aldrich) for the indicated times, and eATP-induced gene
expression changes in IL-1β, IL-6, IL-11, TNF-α, G-CSF, IFN, Mx, and NF-κ B p65
subunit (p65) were quantified by quantitative real-time PCR (qRT-PCR). For this
aim, one microliter of cDNAs from each source was amplified in a MyiQ™ Two-Color
Real-Time PCR Detection System (Bio-Rad) with corresponding primer pairs (Table 1) in a total
volume of 25 μl using SYBR PrimeScript Ex Taq™ II kit (TaKaRa) under the
following conditions: initial denaturation at 95°C for 30 s, 40 cycles at 95°C
for 5 s, 60°C for 30 s, followed by dissociation curve analysis (55°C to 95°C:
increment 0.5°C for 5 s). β–Actin was used as an internal
reference gene. Agarose gel electrophoresis analyses were performed at the end
of each qRT-PCR to validate specificity of amplification. The identities of all
the qRT-PCR products were further verified by DNA sequencing. The results are
expressed as fold changes in the target gene normalized to the reference gene
and as relative to expression in untreated controls. Data are presented as the
means ± SEM from triplicate experiments.