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6 protocols using m1 macrophage generation medium dxf

1

Differentiation of M1 Macrophages from PBMCs

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M1 macrophages were differentiated from freshly obtained PBMC using M1 macrophage Generation Medium DXF according to the manufacturer’s protocol (PromoCell GmbH, Heidelberg, Germany). In brief, freshly isolated PBMCs were seeded out in an appropriate amount of Monocyte Attachment Medium (C-28051, PromoCell GmbH) in a density of 1 million/cm2 and incubated for 1–1.5 hours at 5% CO2, 37°C. Subsequently, the non-adherent cells were removed by three washing steps with warm monocyte attachment medium, the M1 macrophage Generation Medium DXF (C-28055, PromoCell GmbH) was added to the remaining adherent monocytes and the cells were incubated with refreshment of the M1 macrophage Generation Medium DXF every 3 days for 9 days at 5% CO2 and 37°C. At day 10, the differentiated M1-Macrophages were checked by flow cytometry for correct differentiation, transfected as described below and used for the mRNA microarrays and ELISAs.
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2

Monocyte-derived M1 Macrophage Generation

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Monocytes preenriched from PBMC using EasySep™ Kit were seeded as above and differentiated for 7 days in M1 Macrophage Generation Medium DXF (Promocell, Germany) according to the manufacturer's protocol.
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3

Primary Human M1 Macrophage Transport

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Primary human M1 macrophages were obtained from PromoCell (Heidelberg, Germany) in M1-Macrophage Generation Medium DXF supplemented with HEPES at a final concentration of 25 mM. Cell culture plates were hermetically sealed to avoid spillage and contamination during transport. Complete cell culture sets of primary human M1 macrophages were transported in actively tempered (37°C) closed sterile containers in cabin and connected to internal aircraft power systems on board of Lufthansa flights LH464 from Frankfurt to Orlando on March 11th, 2014 and on April 7th, 2014.
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4

Cell Culture and Differentiation Protocols for Macrophage Studies

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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).
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5

Influenza A Virus Infection Model

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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)].
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6

Macrophage Phenotypic Polarization Protocol

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The murine macrophage-like cell line, J774A.1, was obtained from the Health Science Research Resources Bank (Tokyo, Japan) and cultured in Dulbecco's minimal essential medium (DMEM) with 10% fetal bovine serum (FCS). In addition, these cells were treated with 100 ng/mL of lipopolysaccharide (LPS; Wako, Tokyo, Japan) and 20 ng/mL of interferon (IFN) γ (Wako) for M1-like macrophages or 20 ng/mL of IL-4 (R&D Systems, Minneapolis, MN) for M2-like macrophages.
A human macrophage cell line was obtained from PromoCell GmbH (Heidelberg, Germany) and cultured in Monocyte Attachment Medium (PromoCell GmbH). In addition, for their differentiation into M1- and M2-like macrophages, cells were cultured in M1-Macrophage Generation Medium DXF (Promo-Cell GmbH) and M2-Macrophage Generation Medium DXF (PromoCell GmbH), respectively.
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