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2 protocols using human macrophage colony stimulating factor m csf

1

Macrophage Activation by LPS and HMGB1

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Lipopolysaccharide (LPS, E. coli.0111: B4), triton X-114 and human macrophage-colony stimulating factor (M-CSF) were purchased from Sigma (St. Louis, MO, USA). Isopropyl-D-thiogalactopyranoside (IPTG) was from Pierce (Rockford, IL, USA). DNase I and 2xYT medium were obtained from Life Technologies (Grand Island, NY, USA). Hydrogen peroxide (3 %) was from Fisher Scientific (Waltham, MA, USA). Dithiothreitol (DTT, 100 mmol/L) was obtained from Invitrogen (Carlsbad, CA, USA). HMGB1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Shino-Test Corporation (Tokyo, Japan).
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2

Haptoglobin Purification and CD163 Expression

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Human haptoglobin (from pooled human plasma, a mixture of Hp1-1, 2-1, 2-2, Cat #3536), triton X-114, imidazole, human macrophage-colony stimulating factor (M-CSF), acetaminophen and dexamethasone (Cat # D2915) were purchased from Sigma (St. Louis, MO). NHS-activated sepharose 4 fast flow beads were obtained from GE Healthcare (Cat #17-0906-01, Uppsala, Sweden). Trizol reagent was from Life Technologies (Carlsbad, CA). The RevertAid™ First Strand cDNA Synthesis Kit was from Fermentas (Vilnius, Lithuania). SYBR Premix Ex Taq™ II was from TaKaRa Bio Inco (Otsu, Japan). Thioglycollate medium was purchased from Becton Dickinson Co., (Sparks, MD). Primers for QPCR, trypsin-EDTA and carbenicillin were from Invitrogen Inc., (Carlsbad, CA). Isopropyl-D-thiogalactopyranoside (IPTG) was purchased from Pierce (Rockford, IL). Recombinant human CD163 protein, CD163 expression plasmid (SC117495, NM_004244.3) and MegaTran 1.0 transfection reagent were purchased from Origene (Rockville, MD). Antibodies for human heme oxigenase-1 (HO-1, Cat # ab52946) and CD163 (Cat # MCA1853) were from ABcam (Cambridge, MA) and Serotec (Raleigh, NC) respectively. Anti-human CD163-PE and anti-human CD80-PE antibodies were from BioLegend (San Diego, CA). FITC antibody labeling kit is from Thermo Scientific (Rockford, IL). Alanine transaminase (ALT) assay kits were from BIOO Scientific Corp., (Austin, TX).
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