2 mercaptoethanol m3148

Cladophora cellulose was provided by FMC Biopolymer (batch 3095-10; Newark, DE, USA). FIX-rich PCC was provided by National Center for Hematology, Moscow, Russia, as lyophilized powder. Coliphages ΦX174 (ATCC 13706-B1™) and PR772 (BAA-769-B1), and the host bacteria Escherichia coli (Migula) Castellani and Chalmers C (ATCC 13706) and K12 J53-1(R15) [HER 1221] (BAA-769) strains were obtained from ATCC (Manassas, VA, USA). Agar (214530) was obtained from BD (Franklin Lakes, NJ, USA). Tryptone (LP0042B) and yeast extract (Oxoid) (LP0021) were obtained from Thermo Fisher Scientific. Phosphate-buffered saline (P4417), 2-mercaptoethanol (M3148), sodium chloride (S5886), sodium phosphate dibasic (71640) and 2-mercaptoethanol (M3148) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Any kD™ Mini-PROTEAN^® TGX Stain-Free™ protein gels (4568125), tris/glycine/SDS running buffer (1610732), 4x Laemmli Sample Buffer (1610747), and Precision Plus Protein™ unstained protein standards (1610363) were purchased from Bio-Rad (Hercules, CA, USA).

Bone marrow cells were isolated from femurs of 3–6 months old male TG2 null mice and their wild type counterparts. Bone marrow macrophages (BMDMs) were differentiated in DMEM supplemented with 10% FBS (12106C), 2 mM L-glutamine (G7513), 1 mM Na-pyruvate (S8636), 50 μM 2-mercaptoethanol (M3148) and 100 U/ml penicillin/100 μg/ml streptomycin (P4333) all from Sigma-Aldrich and 10% L929 fibroblast conditioned media for 7 days. For metabolic activation, differentiated macrophages were treated with a combination of 30 mM D-glucose (G8270), 10 nM insulin (12643) and 0.4 mM sodium-palmitate (P9767) all from Sigma-Aldrich for 24 h (16). Sodium palmitate was prepared by diluting a 200 mM stock solution in 70% ethanol into 10% fatty acid-free, low-endotoxin BSA (Sigma Aldrich, A8806 adjusted to pH 7.4) to obtain a 5 mM palmitate-BSA stock solution that was filtered using a 0.22-μm low-protein binding filter (Millipore). BSA/ethanol was used in control treatments during the protocol. In some experiments during the 24 h metabolic activation BMDMs were treated with PP2 (Sigma Aldrich, 529573), a reversible ATP-competitive inhibitor of the Src family of protein tyrosine kinases, in 2 µM final concentration or 0.5 mg/ml RGD peptide (Cayman Chemical, 529573).