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Opi media supplement

Manufactured by Merck Group
Sourced in United States

OPI Media Supplement is a laboratory product developed by Merck Group. It is a chemically defined, animal component-free medium supplement designed to support the growth and maintenance of various cell lines in cell culture applications.

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6 protocols using opi media supplement

1

Antibody Purification from Hybridoma Cells

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Hybridoma cells were cultured with hybridoma serum-free medium (Invitrogen) supplemented with OPI Media Supplement (sigma-aldrich). Antibodies were purified from the conditioned culture media with a HiTrap Protein G-Sepharose column (GE Healthcare Life Sciences). The antibodies are in PBS after going through a PD-10 desalting column (GE Healthcare Life Sciences).
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2

Isolation and Culture of Human Monocytes

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Human monocytes were derived from fresh whole blood within 1 hour of venipuncture via double Ficoll (Sigma-Aldrich, St Louis, MO) and Percoll (Sigma-Aldrich) centrifugation as described in Turk et al.18 (link) Monocytes were cultured in MEM (Thermo Fisher Scientific, Waltham, MA) with NEAA (Gibco, Grand Island, NY; Thermo Fisher Scientific), L-glutamine (Gibco, Thermo Fisher Scientific), 1% Pen/Strep (Gibco, Thermo Fisher Scientific), OPI Media Supplement (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific), IL-4 (Sigma-Aldrich; 20ng/ml), and GM-CSF (Sigma-Aldrich; 50ng/ml). Forty-eight–well plates were seeded with 200,000 cells per well to ensure adherence and cultured for 7 days prior to VLCFA exposure.
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3

Analyzing NF-κB Signaling in Cell Cultures

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All cell culture reagents were obtained from Invitrogen (Carlsbad, CA, USA), OPI media supplement and LPS from E. coli (0111:B4) were purchased from Sigma Aldrich (St. Louis, MA, USA). For Western blot analysis, cells were lysed in Pierce M-PER lysis buffer (Rockford, IL, USA) and applied on SDS-Gels with 4x NuPAGE sample buffer (Invitrogen). Nuclear fractions were gained by applying the Nuclear Extract Kit provided by Active Motif (Carlsbad, CA, USA) according to the manufacturer’s protocol. Antibodies against p-IκBα, IκBα, p-NF-κB, NF-κB, p-ERK, ERK, p-p38 as well as p38 were from Cell Signalling (Danvers, MA, USA), whereas the antibody against gp96 was purchased from Stressgen (Ann Arbor, Michigan, USA). The antibodies against β-actin and lamin A/C were from Millipore (Bedford, MA, USA) and BD Biosciences (Allschwil, Switzerland), respectively. Horseradish-peroxidase coupled secondary antibodies were from Santa Cruz. Chemiluminescent substrate ECL plus and films were from GE Healthcare (Chalfont, UK). IL-8 ELISA was obtained from Invitrogen. All other reagents were of analytical grade and acquired commercially.
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4

Culturing Human Monocyte Cell Lines

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Mono Mac 6 cells (DSMZ ACC 124)25 (link), a human monocyte-derived cell line exhibiting a mature monocyte phenotype, 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, Carlsbad, CA, USA), and 1% OPI media supplement (Sigma-Aldrich, St. Louis, MO, USA). THP-1 cells (ATCC TIB-202)29 (link), another human monocyte-derived cell line, were grown and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin. These cells were grown at 37 °C in 5% CO2 in a humidified incubator.
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5

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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6

Culturing Human Neuroblastoma KELLY Cells

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Human neuroblastoma KELLY cells (Sigma-Aldrich, St. Louis, MO, USA) were maintained in RPMI-1640+HEPES (HyClone) as previously described.[7 (link)] Cells were supplemented with 5% FBS, 50mg ml−1 gentamicin, 1% OPI Media Supplement (Sigma-Aldrich), and 1% a,l-glutamine solution (Gibco). All cells were maintained in a 5% CO2 atmosphere at 37°C and EDTA-passaged at 80% confluence.
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