Growth and Differentiation of Magnetic Nano Particle-Loaded Neurons on Magnetic Platforms
Transcrição
Seed 1 million cells in a regular, uncoated 35-millimeter dish. Add the calculated volume of MNP suspension and differentiation medium to the dish to achieve the desired MNP concentration. Mix the cells, MNPs, and differentiation medium, then incubate the dish in a 5% carbon dioxide humidified incubator at 37 degrees Celsius for 24 hours.
Clean the patterned substrate with 70% ethanol, and place it in a 35-millimeter culture dish in the hood. Place a large magnet below the patterned substrate for one minute, then remove it by moving the dish up and away, and taking the magnet out of the hood. Turn on the ultraviolet light for 15 minutes.
To prepare a collagen-coated glass substrate, dilute collagen type I at a 1-to-50 ratio in 30% ethanol. Cover the glass with the solution. Leave the dish uncovered in the hood for four hours until all the solution evaporates. Then, wash it three times in sterile PBS. Remove the cells from the incubator. Centrifuge the cell suspension for 5 minutes at 200 g and discard the supernatant. Resuspend the cells in 1 milliliter of fresh differentiation medium, and count the cells using a hemocytometer.
Seed 105 cells atop the substrate in a 35-millimeter culture dish, and add 2 milliliters of differentiation medium. Incubate the culture in a 5% carbon dioxide humidified incubator at 37 degrees Celsius. After 24 hours, add 1 to 100 fresh Murine beta-NGF to the cells. Renew the differentiation medium and add fresh beta-NGF every two days.
