Eagle s minimal essential medium emem

A variety of cell lines were tested to improve the chances of isolating mammalian viruses, as some viruses replicate in particular cell lines. Cell lines A549 (CCL-185), BHK-21 (CCL-10), HeLa (CCL-2), LLC-MK2 (CCL-7), MDCK, (CCL-34), Mv1 Lu (CCL-64), Neuro-2a (CCL-131), NIH/3 T3 (CRL-1658), and Vero E6 (CRL-1586) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), and propagated as monolayers at 37°C and 5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM) (Mediatech, Inc., Manassas, VA, USA) or Eagle's Minimal Essential Medium (EMEM) (Invitrogen, Carlsbad, CA, USA), as appropriate per cell line. DMEM and EMEM were supplemented with 2 mM L-Alanyl-L-Glutamine (GlutaMAX, Invitrogen, Carlsbad, CA, USA.), antibiotics [PSN; 50 µg/ml penicillin, 50 µg/ml streptomycin, 100 µg/ml neomycin (Invitrogen, Carlsbad, CA, USA)], and 10% (v/v) low IgG, heat-inactivated gamma-irradiated fetal bovine serum (HyClone, Logan, UT, USA). EMEM was also supplemented with sodium pyruvate (Invitrogen Corp.) and non-essential amino acids (Hyclone, Logan, UT, USA).

Emphasis was on the isolation of influenza viruses and genetic analyses thereof, but lesser efforts were nevertheless exerted to isolate other common “culturable” human respiratory viruses to gain insights on the general utility of the VIVAS for virus aerosols. The term “culturable” refers to those viruses that can be isolated and propagated using standard cell lines and methods. To favor isolation of a wide variety of respiratory viruses, concentrated air sample collection media were inoculated onto a variety of readily available (“standard”) cell lines obtained from the American Type Culture Collection (ATCC) for virus isolation attempts, including the following: A549 (CCL-185), HeLa (CCL-2), LLC-MK2 (CCL-7), MDCK (CCL-34), MRC-5 (CCL-171), NCI-H292, and Vero E6 (CRL-1586). Common human respiratory viruses that can be isolated using these cells are shown in Table S1 (T. Bonny and J. Lednicky, unpublished data). All the cell lines were propagated as monolayers at 37°C and 5% CO₂ in advanced Dulbecco’s modified Eagle’s medium (aDMEM) or Eagle’s minimal essential medium (EMEM) (both from Invitrogen, Carlsbad, CA), as appropriate per cell line. Prior to the preparation of seed stocks, each cell line was treated for 3 weeks with plasmocin and verified free of mycoplasma DNA by PCR (37 ).