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These proteins were further purified from cell media by affinity chromatography using an M2 (anti-FLAG) affinity column (Sigma-Aldrich), followed by gel filtration chromatography on a Sephacryl SHR100 column (Amersham, GE Healthcare Life Sciences) to 95% or greater purity, confirmed by SDS-PAGE (data not shown), with a yield of 3 to 5 mg/l medium. Stable Drosophila cell lines expressing uPA-T or PLT/uPA-T were used to produce recombinant proteins. Samples were centrifuged at 14,000 rpm, for 30 min at 4°C, and equal amounts of proteins were used for PAGE and western blot analysis. These mice were genotyped by PCR analysis of tail genomic DNA using previously described primer pairs and reaction conditions. Data has been generated with t he help of GSA Content Generator Demov ersion ! Measurement of tail lymphatic flow. Flow cytometry. buy nembutal pwoder near me (0.5 ml) was incubated with 10 ml red blood cell lysis buffer for 10 minutes at room temperature and passed through a 50-μm mesh (Falcon; BD Biosciences), yielding single-cell suspensions. CD11b-expressing cells in kidney single-cell suspensions or cells isolated from the peritoneal cavity were enriched using mouse CD11b Microbeads and MACS. The fixed kidney and skin were dehydrated through a graded series of ethanols, embedded in paraffin, sectioned (4 μm), and mounted on glass slides. Isolation of kidney and peritoneal monocytes/macrophages/dendritic cells. RNA isolation and qRT-PCR. Quantitative reverse-transcriptase PCR (qRT-PCR) was performed using TaqMan real-time PCR (7900HT, Applied Biosystems). After immunostaining, cells were analyzed immediately on a FACSCanto II cytomer with DIVA software (BD), and off-line list mode data analysis was performed using Winlist software from Verity Software House. 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