Catalog 48300

Meniscus cells were isolated from the outer or inner zones (approximately outer one-third and inner two-thirds respectively) of the medial meniscus (N=6) by sequential digestion of minced tissue with 0.5% (w/v) pronase (SKU 53702; EMD Millipore Corp., Temecula, CA) digestion for one hour and 0.2% (w/v) collagenase type I (Catalog # LS004197; Worthington Biochemical Corp., Lakewood, NJ) digestion for 16 hours in DMEM-HG with 10x antibiotic/antimycotic and 10% FBS²³ . Isolated cells were seeded at 5 million cells per 10 cm tissue culture plate and maintained in complete culture media. Media was replaced every 2-3 days and cells were passaged at 90% confluence (5-6 days for passage 1 and then 2 days for subsequent passages). RNA was extracted by column purification (Catalog # 48300; Norgen Biotek Corp.) at the time of the first, second, and third passage (p1, p2, and p3).

Explants were pulverized in TRIzol (Catalog # 15596026; Life Technologies, Carlsbad, CA) under liquid nitrogen using a freezer mill (Model 6875; SPEX SamplePrep, Metuchen, NJ). Then RNA was extracted by TRIzol/choloroform separation following the manufacturer’s protocol and column purified (Catalog # 48300; Norgen Biotek Corp., Thorold, Canada).