Penicilin streptomycin

12-well cell culture plates (TPP) were pre-coated with different amounts of chimeric mouse DLL1-Fc protein or Fc fragment alone (both from R&D) dissolved in PBS, at RT for 3hr. Single cell suspensions were prepared from the bone marrows of Cx3cr1^gfp/+ mice, stained for CD11b and Ly6C and GFP⁺CD11b⁺Ly6C^hi monocytes were sorted as described above. 1.6x10⁵ cells were plated in pre-coated wells, in RPMI-1640 medium (BioCell), supplemented with 10%FCS (Biochrom), 1% Glutamax (Gibco) and 1% Penicilin-Streptomycin (Biochrom), in the presence of different concentrations of recombinant mouse M-CSF (Peprotech). After 48hr of culture, medium was supplemented with the new dose of M-CSF, equal to initial amounts. Finally, cells were collected after 72hr of culture and used for flow cytometry analysis or RNA isolation. To quantify proliferation, BrdU (Sigma) was added to culture medium at a 10μM final concentration, during the last 16hr of 72hr culture and incorporation was detected by flow cytometry, using BrdU flow kit (BD Pharmingen), according to manufacturer’s instructions.

All chemicals including cell culture medium (Dulbecco´s modified Eagle´s medium (DMEM)) as well as the catalase-polyethylene glycol (PEG-catalase) were obtained from Sigma or Merck (Darmstadt, Germany) unless otherwise stated. The fetal bovine serum was from Pan Biotech (Aidenbach, Germany). The Protein Assay Kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories (Feldkirchen, Germany). The enhanced chemiluminescence system (SuperSignal West Pico/Femto Maximum Sensitivity Substrate) was supplied by Pierce (Fisher Scientific, Schwerte, Germany). Penicilin/Streptomycin was obtained from Biochrom (Berlin, Germany) and Glutamax from Gibco (Darmstadt, Germany). MitoTEMPO was purchased from Enzo Life Sciences (Lausen, Schweiz). MitoSOX Red and MitoTRACKER Green FM were obtained from Fisher Scientific (Schwerte, Germany). Beta-actin antibody was purchased from Cell Signaling (Massachusetts, USA). The polyclonal rabbit α-hapten antibody directed against dimedone tagged sulfenic acids was kindly provided from K.S. Carroll [35 (link)]. TOM-20 antibody was purchased from Santa Cruz Biotechnology (Dallas, USA). DMSO was obtained from Roth (Karlsruhe, Germany). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG from Dianova (Hamburg, Germany) and HRP conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used as a secondary antibody.

All chemicals including cell culture medium (Dulbecco´s modified Eagle´s medium (DMEM)) were obtained from Sigma or Merck (Darmstadt, Germany) unless otherwise stated. The fetal bovine serum was from Pan Biotech (Aidenbach, Germany). The Protein Assay Kit (Bio-Rad DC, detergent compatible) was from Bio-Rad Laboratories (Feldkirchen, Germany). The enhanced chemiluminescence system (SuperSignal West Pico/Femto Maximum Sensitivity Substrate) was supplied by Pierce (Fisher Scientific, Schwerte, Germany). Penicilin/Streptomycin was obtained from Biochrom (Berlin, Germany) and Glutamax from Gibco (Darmstadt, Germany). Heptelidic Acid was purchased from Cayman Chemical Company (Distributor Biomol GmbH, Hamburg, Germany). Beta-actin antibody was purchased from Cell Signaling (Massachusetts, USA). DMSO was obtained from Roth (Karlsruhe, Germany). Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG from Dianova (Hamburg, Germany) and HRP conjugated rabbit anti-mouse IgG from Dako (Glostrup, Denmark) were used as a secondary antibody. The Sulfo Biotics Protein Redox Monitoring Kit was delivered by Dojindo EU GmbH, Munich, Germany.