The frozen swine kidney cells stored in each cryotube were rapidly thawed at 37 °C and split into three or five T-75 flasks. After being cultured for 7–10 days, a mixed monolayer cell sheet formed, and macrophage-like cells began to actively proliferate on the cell sheet. The proliferating macrophage-like cells were loosely attached to the cell sheet, and large numbers of macrophage-like cells (1×105–1.5×106 cells/T-75 flask) were harvested from the culture supernatant by centrifugation (1500 rpm for 5 min) every 3–4 days for 1–2 months. Immunocytochemical

analyses were performed as described previously [10] and [11]. Primary antibodies against cytokeratin this website 18 (CK18; Millipore Co., Billerica, MA), cytokeratin

PF-02341066 chemical structure 19 (CK19; Progen, Heidelberg, Germany), α-smooth muscle actin (SMA; Progen), macrophage scavenger receptor MSR-A:CD204 (KT022; TransGenic, Inc., Kumamoto, Japan), Iba 1 (Wako Pure Chemical Industries, Ltd, Osaka, Japan), and CD172a (VMRD, Inc., Pullman, WA) were used. Macrophages were also isolated from a mixed primary culture of swine liver tissue, as described previously [10]. Mouse kidney macrophages were isolated from a mixed primary culture of C57BL/6 mice kidney cells according to the protocol used to isolate macrophages from the swine kidney tissue, before being immortalized by retroviral transduction of human c-myc, as described in our previous studies [12]. The clonal macrophage cell line (KM-1) was established and routinely cultured with growth medium. RT-PCR analyzes were performed as described previously [13], with minor learn more modifications. The following oligonucleotide primers were used: mouse P2X7R (NM011027): sense, 5′-GACAAACAAAGTCACCCGGAT-3′, and antisense, 5′-CGCTCACCAAAGCAAAGCTAAT-3′; swine P2X7R (XM001926804): sense, 5′-GACAAACAAAGTCACCCGGAT-3′, and antisense, 5′-CTTGTCACTCACCAAAGCAAAG-3′; swine glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

(NM001206359): sense, 5′-TCACCAGGGCTGCTTTTAAC-3′, and antisense, 5′-GATCTCGCTCCTGGAAGAT-3′. The amplified DNA fragments derived from mouse P2X7R, swine P2X7R, and swine GAPDH mRNA were 101, 106, and 192 bp (bp) long, respectively. A mouse β-actin primer (#G5740, Promega, Madison, WI) was also used, and the resultant 285 bp DNA fragment was amplified. The cells were re-suspended in HEPES-buffered salt solution (HBSS; 145 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 20 mM HEPES, 10 mM glucose, and 0.01% BSA; pH 7.4), and the [Ca2+]i was measured at 30 °C by monitoring fura-2 fluorescence at 500 nm [excitation wavelengths of 340 (F340) and 380 nm (F380)] as described previously [12] and [13]. The ratio of the fluorescence intensities observed at the two above mentioned wavelengths (F340/F380) was used as an indicator of the [Ca2+]i. One milliliter of cell suspension containing 5×105 cells was used in each set of experiments.