M1 macrophage generation medium dxf

The RAW264.7 (ATCC TIB-71) [18 (link)] and THP-1 cells (ATCC TIB-202) [20 (link)] were grown and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin; and Mono Mac 6 cells (DSMZ ACC 124) [19 (link)] were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 1% non-essential amino acids (Life Technologies), and 1% OPI media supplement (Sigma-Aldrich). These cells were grown at 37°C in 5% CO2 in a humidified incubator. To differentiate THP-1 cells into macrophages, THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) at the concentration of 100 nM for 72 hours. The cells were incubated in growth medium without PMA for an additional 24 hours, and used in the study. Normal human monocytes were purchased from commercially available products (Promocell, Heidelberg, Germany). The cells were differentiated into macrophages using a commercially available medium (M1-Macrophage Generation Medium DXF; Promocell) in accordance with the manufacturer's protocol, and used in the study. The procedure for preparation of the purified AP-PG from A. pullulans cultured fluid was described elsewhere [15 (link)]. Curdlan derived from Alcaligenes faecalis was obtained from commercially available products (Sigma-Aldrich).

Primary human lung fibroblasts (IMR-90s, Coriell Institute for Medical Research, Camden, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich, Taufkirchen, Germany) with 10 % fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany), and 1 % penicillin/streptomycin (P/S, Lonza, Basel, Schweiz).
Human monocyte-derived macrophages (hMDM) were isolated from healthy donors by using Histopaque (Sigma Aldrich) and separated by density gradient centrifugation, plated in monocyte attachment medium (Promocell, Heidelberg, Germany) for 2 h and cultivated in M1-Macrophage Generation Medium DXF (Promocell) for 7 d.
Madin-Darby canine kidney (MDCK) cells were cultured in Eagle minimum essential medium (EMEM, Sigma Aldrich) with 10 % FBS and 1 % P/S.
In vitro infections were performed using influenza A virus/IAV/H1N1/Puerto Rico/8/1934 (PR8). For the in vivo model the mouse-adapted strain influenza A virus/IAV/H1N1/vi0084/2016 was used. Ex vivo infections utilized the mouse-adapted H1N1 influenza A virus strain HA-G222-mpJena/5258 [15 (link)]. To identify plaque-forming units (PFU), standard plaque assay was performed as described previously [16 (link)].