Lipofectamine ltx with plus

The mutant MeV library was rescued using standard protocols with modifications (Krumm et al., 2013 (link); Radecke et al., 1995 (link)). 5μg of MeV genome and four helper plasmids were transfected into 70% confluent BSR-T7 cells in 6-well dishes using the Lipofectamine LTX with Plus (Invitrogen) reagent. A total of five 6-well plates were used for rescuing the viral libraries. At 16 hours post-transfection, the media was replaced with fresh DMEM. Five days post-transfection, the transfected cells were scraped into their media, pooled, and then co-cultured with 60% confluent A549s in 15 cm dishes. After 3 days of co-culture, the cells were again scraped into their media freeze thawed at −80°C and pelleted by centrifugation 4000xg for 5 m ins. The clarified supernatant was used to infect 70% confluent A549s in 15 cm dishes. After 3 days (sufficient time to allow high levels of viral replication to occur), the infected cells were scraped into their media. Half of the cells were pelleted, lysed in 3 mL TRIzol, and frozen at −80°C. The remaining cells were freez e thawed and pelleted by centrifugation (4000xg for 5 mins). The clarified supernatant was again used to infect 70% confluent A549s for a second passage of 3 days in 15 cm dishes. After 3 days, the supernatant was removed and the infected cells were lysed in 6 mL TRIzol and frozen at −80°C.

Flp-In 293 cell lines that stably express ^V5hDuoxA1α or ^V5hDuoxA2 were previously described by Morand, et al. [7 (link)]. Briefly, cells were cultured in minimum essential medium-α supplemented with 10% fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml hygromycin B (Life Technologies, Carlsbad, CA, USA) in a 5 % humidified CO₂ incubator at 37 °C. These lines were regularly assayed by Western blotting with anti-V5 to monitor DuoxA protein expression. Cells were transiently transfected with pcDNA5/FRT plasmid encoding human ^HADUOX2, V674G/V674A/V674L/V674T ^HADUOX2, ^HADUOX1 or V670G ^HADUOX1 cDNAs using the FuGene^® 6 (Roche, Indianapolis, USA) or Lipofectamine^® LTX with Plus™ (Life Technologies) transfection reagents according to the manufacturer's instructions. The cells were typically seeded in 6-well plates and transfected with 1-2 ug of plasmid DNA 24 hours later upon reaching densities of ∼70% confluence. In some experiments transfection efficiencies were confirmed by including EGFP reporter plasmid (1:5 relative to Duox plasmid) or by determining plasmid-encoded Duox transcript copy numbers (relative to GAPDH) from reverse transcription/real time-PCR reactions by methods described earlier [36 ], using the SYBR Green PCR Mix (Invitrogen) and an ABI Prism 7500 RT-PCR System (Applied Biosystems/Life Technologies).