Vascular smooth muscle cell contractility assay

The ability of cells to contract a collagen gel matrix was determined using a 96-well collagen gel contraction assay using standard protocol[40]. Aortic VSMCs isolated from Apoe^-/- or Notch1^+/-;Apoe^-/- mice were first cultured in a 6-well plate and treated with or without 10 ng/ml CTGF for 72 hours. Cells were then trypsinized, counted, and suspended in a collagen solution at a concentration of 6x10⁵ cells/ml. This collagen solution contained the following: 43% (v/v) sterile PBS (Hyclone), 34% (v/v) 3 mg/ml rat tail collagen (BD Biosciences), 13.6% (v/v) serum-free DMEM (Hyclone), 7.6% (v/v) 10x Medium 199 (GIBCO), and 1% (v/v) FBS (Mediatech). The solution was brought to neutral pH (~7.0) by addition of NaOH using the phenol red indicator in the DMEM. Then, 70 μl of this collagen+cell solution was loaded into the wells of a 96-well plate and incubated at 37°C for 30 minutes to allow collagen to polymerize. A total of 140 μl of DMEM with 2% FBS was then added and the wells were incubated for 48 hours. The gels were then gently released from attachment to walls of the wells by Dumont #55 Forceps and 0 hour images were taken. The media was then replaced with 10% FBS DMEM to induce contraction and incubated for further 2 hours. After 2 hours, another image was taken of each well to determine the amount of gel contraction. Each treatment group was represented by 6 replicates in each experiment, and repeated in 3 independent experiments. The images were analyzed using ImageJ software[41] to measure the gel area at each time-point, from which the percentage decrease of gel area was determined[42].