Ccl 185

Human lung adenocarcinoma cells (A549, ATCC^® CCL185^TM) and Madin-Darby canine kidney cells (MDCK II, ATCC^® CRL2936^TM) were cultured in IMDM medium. Human embryonic kidney cells expressing the SV40 large T-antigen (HEK 293T, ATCC^® CRL-3216^TM) were maintained in DMEM Medium. Following wild type influenza A virus strains were used: A/Mississippi/3/2001 (H1N1), A/Perth/265/2009 (H1N1pdm09), A/Puerto Rico/8/34 (H1N1), and A/Victoria/03/75 (H3N2).

The human tumorigenic lung epithelial cell line A549 was purchased from the ATCC (CCL-185™). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 1% penicillin-streptomycin solution and incubated at 37 °C in a humidified atmosphere with 5% CO₂.
For assays, 10⁴ cells were added to each well of a 96 flat-bottom microplate and kept in an incubator at 37 °C and 5% CO₂ for 24 h. At the end of the incubation, increasing concentrations of antimicrobial agents (5, 10, 20, 50, 75 and 100 mg/L) were added to the wells and incubated for 24 h. The dye MTT was used to determine the cytotoxicities of the antimicrobial agents on the A549 cell line [18 (link)]. Experiments were repeated at least three times. The 50% inhibitory concentrations (IC₅₀) were calculated by using GraphPad Prism version 8 (San Diego, CA, USA).

Tetrachloroauric(III) acid
trihydrate (HAuCl₄·3H₂O), citric acid (CA),
sodium tricitrate dihydrate, and ethylene diamine (EDA) were obtained
from Sigma-Aldrich and used without further purification. Prior to
experiments, all glassware was cleaned with aqua regia and washed
with distilled water. Adenocarcinoma human alveolar basal epithelial
cells (A549, CCL-185, ATCC) and HDF cells (HDFa, PCS-201-012, ATCC)
cell lines were purchased from the American Type Culture Collection.

Human embryonic kidney 293T (Clontech; 632180), human lung carcinoma A549 (ATCC; CCL-185), and human hepatoma Huh7.5 (Apath) cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen; 10-013-CV); African green monkey kidney Vero (ATCC; CCL-81) and human lung epidermoid carcinoma Calu-1 (ATCC; HTB-54) cells were cultured in Eagle’s minimum essential medium (ATCC; 30-2003) and McCoy’s 5a medium Modified (ATCC; 30-2007), respectively. All of the media were supplemented with 10% fetal bovine serum (FBS) (Invitrogen; 10082147) and 1% penicillin/streptomycin. Cells were grown at 37 °C with 5% CO₂.

A549 human lung carcinoma epithelial cells (ATCC^® CCL-185™), HMEC-1 human microvascular endothelial cells (ATCC^® CRL-3243), THP-1 human monocytic cells (ATCC^® TIB-202™) and Vero E6 green monkey epithelial kidney cells (ATCC CRL-1586) were cultured under standard conditions in 24-well cell culture plates. A549 cells and Vero E6 cells were cultivated in DMEM containing 10% FCS and 2 mM L-Glutamine. HMEC-1 cells were cultivated in MCDB131 medium containing 10% FCS, 10 mM L-Glutamine, 10 ng/mL epidermal growth factor (EGF) and 1 µg/mL Hydrocortisone. THP-1 cells were cultivated in RPMI medium containing 10% FCS and 2 mM L-Glutamine. For differentiation into a macrophage-like phenotype, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA) at 100 ng/mL for 72 h.

A549 (ATCC^®, CCL-185™), RPE (ATCC, CRL-2302), Vero (ATCC^®, CCL-81™), and derived cell lines were maintained in DMEM-F12 (GIBCO, 12400-024) containing 5% fetal bovine serum (FBS – GIBCO, 12657-029) supplemented with 1 U/mL penicillin/streptomycin (Sigma, P4333). C6/36 (ATCC^®, CRL-1660™) cells were maintained at 28 °C, in L-15 (Sigma, L4386) containing 5% FBS supplemented with 0.26% tryptose phosphate broth (GIBCO, 18050-39), and 1 U/mL penicillin/streptomycin. Both cell lines were routinely tested for mycoplasma contamination. Cells were treated with 0.5 or 1 µM of NU7441 (BioGems, 5039598), 100 ng/mL human TNF (Peprotech, 300-01A), and 3 µM etoposide (Sigma, E1383).