Medium 199

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	Supplement medium with 15% fetal bovine serum with penicillin [100 U/mL], streptomycin [100 U/mL], and amphotericin [0.27 μg/mL]

Protocol tips
Supplement medium with 15% fetal bovine serum with penicillin [100 U/mL], streptomycin [100 U/mL], and amphotericin [0.27 μg/mL]

Publication protocol

Vascular smooth muscle cells were isolated from medial layer explants of thoracic aorta from 3-month old female pigs and were cultured in standard growth medium ([GM] M-199/15% fetal bovine serum with penicillin [100 U/mL], streptomycin [100 U/mL], and amphotericin [0.27 μg/mL]). Medium was replaced every 2–3 days. The cellular phenotype of all cells examined was confirmed by positive immunofluorescence staining with antibodies against α-smooth muscle actin as well as smooth muscle myosin heavy chain-2. Stocks of cells were stored in liquid nitrogen, and cells between passages three and six were used for study. Each experiment was initiated by seeding cells in GM into 24-well culture plates and in 4-chamber slides at 1.3×104 cells/cm2. Cells were cultured until nearly confluent (day 0), then divided into three experimental groups differing only in treatment: 1) media alone, no treatment (control); 2) CNPs, ten nephelometric turbidity units per cm2; or 3) 2 mM P as a positive control for calcification.Cells were cultured for up to an additional 28 days before harvesting.

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