Human peripheral mononuclear cells (PBMCs) were isolated from buffy coats (DRK-Blutspendedienst Hessen GmbH, 506838) by passage over a Leukocyte Separation Medium (PAA, J15-004) gradient. The donors were healthy German adults without known exposure to Leishmania parasites. After separation, PBMCs were washed and resuspended in complete medium (CM), consisting out of RPMI 1640 (R0883) supplemented with 10% heat inactivated fetal calf serum (F7524), 50 μM β-mercaptoethanol (M6250) (all from Sigma Aldrich), 2 mM L -glutamine (K 0282), 100 U/ml penicillin and 100 μg/ml streptomycin (A 2213), 10 mM HEPES (L 1613) (all from Biochrom). Monocytes were isolated by exploiting their ability to adhere to plastic, or by CD14 positive selection (Miltenyi, 130-050-201). Nonadherent or CD14-negative cells were collected and frozen in CM containing 40% fetal calf serum and 10% DMSO for lymphocyte proliferation assays. Monocytes were differentiated into human monocyte derived macrophages by addition of 10 ng/ml recombinant human macrophages colony stimulating factor (M-CSF) (R&D, 216-MC), for a period of 5 to 7 d at 37°C, 5% CO2.
β mercaptoethanol m6250
Manufactured by Merck Group
Sourced in United States
β-mercaptoethanol (M6250) is a reducing agent commonly used in biochemical and molecular biology applications. It functions by breaking disulfide bonds in proteins, facilitating the denaturation and analysis of protein structures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Harvard Bioscience (1 mentions)
L glutamine k0282, by Harvard Bioscience (1 mentions)
Streptomycin a2213, by Harvard Bioscience (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
M csf, by R&D Systems (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti nrf2, by Cell Signaling Technology (1 mentions)
Anti grx1, by Abcam (1 mentions)
Salvianolic acid b, by Merck Group (1 mentions)
2 protocols using β mercaptoethanol m6250
1
Isolation and Differentiation of Human PBMCs
2
Salvianolic acid B modulates cellular redox
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Salvianolic acid B was purchased from Sigma-Aldrich (49724, St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM; D5546), fetal bovine serum (FBS; F2442), penicillin/streptomycin (P4333), and 0.25% trypsin (T4049) and other cell culture reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen peroxide (216763), 2-hydroxyethyl disulfide (HEDS; 380474), glutathione (GSH; 1294820), glutathione reductase (GR; 10105678001), β-nicotinamide adenine dinucleotide phosphate-reduced tetra(cyclohexylammonium) salt (NADPH; N5130), and β-mercaptoethanol (M6250) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies including anti-Grx1 (Abcam, Cambridge, UK, ab45953), anti-Grx2 (Abcam, ab191292), anti-PSSG (Virogen, London, UK, 101-A-100), anti-Nrf2 (Cell Signaling, Beverly, MA, USA, #12721), anti-GAPDH (Santa Cruz, CA, USA, sc-32233) antibodies, and horseradish peroxidase-conjugated secondary antibodies (Santa Cruz, sc2061, sc2060, sc2030) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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