Reverse Genetics Protocols for Influenza A H1N1pdm09 and Pneumococcus Strains

MDCK (Madin-Darby canine kidney, obtained from ATCC) were grown at 37°C in 5% CO₂ in MEM medium (Sigma) containing 5% Fetal Bovine Serum (FBS, HyClone), penicillin/streptomycin, and L-glutamine. Reverse genetics plasmids of A/California/07/2009 were a generous gift from Dr Jesse Bloom (Fred Hutch Cancer Research Center, Seattle) and were rescued as previously described (Lakdawala et al. 2011 (link)). The viral titers were determined by tissue culture infectious dose 50 (TCID₅₀) using the endpoint titration method on MDCK cells for H1N1pdm09. The bacterial strains used in this study were graciously provided by Dr Hasan Yesilkaya (wild type Spn serotype 2 strain D39; Andrew et al. 2018 (link)) and Dr Jason Rosch (wild type Spn serotype 19F strain BHN97; Rowe et al. 2019 (link)). Bacteria were grown from frozen stocks by streaking on TSA-II agar plates supplemented with 5% sheep blood (BD). Cultures were generated in fresh Columbia broth (Thermo Fisher) and incubated at 37°C and 5% CO₂ without shaking. Pneumococci in nasal washes and tissues were determined by plating serial dilutions onto blood agar plates incubated at 37°C overnight.