Nunc cell culture tubes

76 PCR-positive swabs were further processed for virus isolation. Madin-Darby canine kidney cells (London line, obtained from WHO CC World Influenza Centre, London) were used for isolation of influenza A viruses (IAV) from rRT-PCR positive samples. Briefly, cells were seeded on Nunc cell culture tubes (Nunc, Thermo Fisher Scientific) 1 day before inoculation to form a 90–95% confluent monolayer. Cells were washed twice with MEM media containing 2 μg of TPCK-trypsin and penicillin-streptomycin (10,000 units and 10 mg/ml, respectively), and 200 μl of virus-containing media was inoculated into each tube. Tubes were kept at 36°C for 40 min to allow virus absorption. Then 1.8 ml of virus growth media was added [MEM containing 2 μg of TPCK-trypsin and penicillin-streptomycin, bovine albumin fraction V (2.6 ml per 100 ml, Sigma Aldrich), HEPES buffer (1.6 ml, Sigma-Aldrich)]. Tubes were kept for 3–6 days at 36°C and monitored daily for progression of CPE. Once CPE was detected, the tubes were frozen at −80°C, thawed, and the hemagglutination assay with human (group O) red blood cell suspension (0.75%) was performed to determine viral titers. Isolated viruses were further sequenced.

In a set of 3 independent experiments, cells were seeded in quadruplicate in flat-sided Thermo Fisher Scientific™ Nunc™ Cell Culture Tubes at an initial seeding density of 50 k cells/ml of serum free culture medium. Cells were treated as described above. The conditioned culture medium was exchanged daily, and the cells for each group were harvested for counting at 24-hour intervals over a four-day period. An MTS assay was also used to measure proliferation and viability. Clear 96 well plates were seeded with 5,000 cells/well in serum free conditions. Cells were treated as described above, and conditioned medium was refreshed daily. Cell proliferation was measured with the CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) kit following manufacturer instructions (Promega, Madison, WI) at 24 and 48 hours as measured by the Synergy HT Plate Reader (Biotek, Winooski, VT).