Platelet derived growth factor pdgf bb

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), antibody to β-actin, and lipopolysaccharide (LPS) were purchased from Sigma (St Louis, MO, USA). The antibodies to phosphorylated ERK (pERK1/2), ERK1/2, pJNK, JNK, pFAK, and FAK were purchased from Cell Signaling Technology (Beverly, MA, USA), antibodies to RhoA, Cdc42, and GAPDH were purchased from Santa Cruz (Santa Cruz, CA, USA), and antibody to MMP-9 was obtained from Millipore (Billerica, MA, USA). Platelet-derived growth factor (PDGF-BB) and tumor necrosis factor alpha (TNF-α) were obtained from R & D systems (Minneapolis, USA). Nitrocellulose membrane and chemiluminescent for Western blotting were purchased from Bio-Rad (Richmond, CA, USA) and IMEGENEX (San Diego, CA, USA), respectively. All solvents, chemicals, and reagents were of analytical grade and purchased from Sigma-Aldrich unless otherwise specified.

Rat SKPs (rSKPs) were isolated from the back skin of P30 male green fluorescent protein (GFP)‐expressing Sprague Dawley rats (Sadaaki Takagi Japan SLC, Hamamatsu, Japan, http://www.jslc.co.jp) after an overdose of sodium pentobarbital (27.3 mg/kg, intraperitoneal injection), according to previously described protocols 21. Primary dermal cells were grown in basic fibroblast growth factor (40 ng/ml; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), platelet‐derived growth factor (PDGF)‐BB (25 ng/ml; R&D Systems, Minneapolis, MN, http://www.rndsystems.com), B27 supplement (2%; Thermo Fisher Scientific Life Sciences, Waltham, MA, http://www.thermofisher.com) and penicillin/streptomycin (1%; Thermo Fisher Scientific Life Sciences) in Dulbecco's modified Eagle's medium (DMEM) low glucose/F12 (3:1; Thermo Fisher Scientific Life Sciences). SKPs were plated at 30,0000 cells per milliliter in suspension culture flasks (Falcon). Following primary colony formation, SKPs were dissociated to single cells using collagenase digestion (2 mg/ml; Worthington Biochemical Corp., Lakewood, NJ, http://www.worthington‐biochem.com) and replated at 30,000 cells per milliliter. SKPs were passaged three or four times in static culture before being introduced to the bioreactor expansion to obtain sufficient numbers of cells to seed into the 500‐ml bioreactors at 30,000 cells per milliliter.