Lipoprotein deficient serum from fetal calf

Human tendon cells were isolated from healthy human hamstring (semitendinosus) tendons (excess anterior cruciate ligament autograft material) as previously described [20 (link)]. Male (N = 2; age 28 ± 1.4) and female (N = 2; age 37 ± 5.7) donors provided written informed consent and the protocol was approved by University of British Columbia Clinical Research Ethics Board (certificate number H10-00220). Equal number of females and males were used in each experiment to exclude the effect of sex as a factor. The tendon cells were dissociated from the tissue using mechanical (chopping with surgical scissors) enzymatic (collagenase) methods. To expand the tendon cell culture, the isolated tendon cells were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 2 mm L-glutamine, and 1x Antibiotic/Antimycotic solution in a humidified incubator containing 5% CO2 at 37 °C. Then, 10% calf serum was replaced with 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) before and during incubation with oxLDL. Recombinant TGF-β protein (PeproTech, USA, #100 − 21) at concentration of 5ng/ml was added during the incubation with oxLDL.

MafB/c-Maf double deficient (Maf-DKO) macrophages were a kind gift from Dr. Michael H. Sieweke (Center d'Immunologie de Marseille-Luminy). Maf-DKO cells were grown in DMEM containing 20% L929-conditioned medium and 10% FCS. BMDM were differentiated from bone marrow cells obtained from C57BL6 mice in the presence of DMEM containing 20% L929-conditioned medium and 10% FCS. Maf-DKO cells were predominantly octaploid (8c) whereas BMDM were diploid (2c) and octaploid (8c) as determined by DNA quantification with DAPI staining (data not shown). For experiments, cells were incubated for 24 h at 37°C and 5% CO₂ in low glucose, low glutamine medium (henceforth LGLG medium) which contained DMEM, 44 mM sodium bicarbonate, 10% FCS, glucose (4.8 mM), and glutamine (0.8 mM) with or without IFNγ 10 ng/mL or IL-4 10 ng/mL (both from Preprotec), and C75, etomoxir, triacsin, oligomycin, DETA/NO, or SEITU (all from Sigma) at the indicated concentrations. In a set of experiments, FCS was replaced with delipidated serum (Lipoprotein deficient serum from fetal calf, Sigma) plus a 1:100 dilution of lipid mixture (Lipid mixture 1, Sigma) containing cholesterol, and arachidonic, linoleic, linolenic, myristic, oleic, palmitic, and stearic acids.

BATs were prepared as previously described [21 (link)] with some modifications. Human tendon cells were seeded in the mixture of Purecol EZ Gel (Sigma-Aldrich, USA, # 5074), 5× DMEM, and 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) with or without 50 µg/ml oxLDL and 5 ng/ml TGF-β. The cells were suspended in the media and other components. Then they were mixed with the neutralized Purecol EZ Gel. The mixture was pipetted between the anchor stems of untreated Tissue Train plate (Flexcell International Corp., USA, #TT-5001U) in each well. After setting the BATs in the incubator, 3ml 1x DMEM media with or without oxLDL and TGF-β was added to each well and the plate was incubated for 72 h (3 days) as the changes in collagen gel by the seeded cells are normally evident within 3 days [22 (link), 23 (link)]. The BATs were then scanned using an image scanner and the images were analyzed in ImageJ to measure the diameter of the BATs.