After incubation at 37 °C for 30 min, the supernatants were collected, and the cells were lysed with 200 µl ice-cold lysis buffer, before IL-1β, P2X7R or actin was detected using immunoblotting, as described previously [14]. The target protein was revealed using ImmunoStar LD (Wako) and detected using a c-Digit Blot Scanner (LI-COR, Inc., Lincoln, NE). YO-PRO-1 or propidium iodide (PI) dye uptake was monitored in live cells cultured in HBSS at 37 °C using a fluorescence microscope and time-lapse

recording, as described previously [14]. Immunostaining demonstrated that almost all of the cells isolated from the mixed primary culture of swine kidney tissue were positive for macrophage markers Imatinib ic50 (KT022, Iba-1, and CD172a), but negative for epithelial (CK18 and CK19) and mesenchymal (SMA) cell markers (Fig. 1A). The proportions of contaminating epithelial and mesenchymal cells High Content Screening comprised less than 1% by cell counting after immunostaining, suggesting that the purity of the macrophages was more than 99%. Semi-quantitative RT-PCR analyses were also performed to investigate the expression of P2X7R. In the KM-1 cells, an amplified

101-bp DNA fragment derived from mouse P2X7R mRNA was detected after 35 and 40 PCR cycles, but not after 30 PCR cycles (Fig. 1B). Similarly, an amplified 106-bp DNA fragment derived from swine P2X7R mRNA was detected in the swine kidney macrophages after 35 and 40 PCR cycles (Fig. 1B). These findings suggest that

the expression level of P2X7R mRNA in swine kidney macrophages is comparable to that seen in KM-1 cells. Furthermore, the expression of P2X7R protein in KM-1 cells or swine kidney macrophages was confirmed by immunoblotting using two different anti-P2X7R antibodies (Fig. 1C). Anti-P2X7R Alomone antibody is known to react with mouse P2X7R despite a 90% homology with its epitope peptide [15]. However, this antibody failed to recognize swine P2X7R, possibly due to the lower (75%) sequence homology with the epitope peptide. Conversely, anti-P2X7R Covalab antibody recognized swine P2X7R (100% identical with its epitope peptide), while did not recognize mouse P2X7R (93% identical). Although the reason why Covalab antibody did Clomifene not react with mouse P2X7R in our experimental conditions is unclear, this may be due to the use of relative lower antibody concentration (0.5 µg/ml) for immunoblotting. Further experiments are required to optimize the reaction conditions for the detection of mouse P2X7R by immunoblotting with Covalab antibody. Although several pro-inflammatory stimuli are reported to modulate P2X7R expression [16], LPS-priming did not affect the protein expression level of P2X7R in KM-1 cells and swine kidney macrophages (Fig. 1C).