Generating Neural Progenitors from Mouse Embryonic Stem Cells by the Hanging Drop Method
Transcripción
Begin by setting up the hanging drop cell culture. For a 10-centimeter cell culture plate, adjust the concentration to 500 cells per 20 microliters of differentiation medium. Prepare enough cell suspension for 50 20-microliter droplets. Use a micropipette or a repeater pipette to place 20-microliter droplets of cell suspension onto the lid of a tissue culture plate, making sure that the droplets aren't too close together to prevent them from merging.
Fill the plate with 5 to 10 milliliters of PBS, and carefully put the lid back on the plate. Incubate the culture in the 37 degrees Celsius incubator. After two days, collect the droplets from the lid, and place them in a 10-centimeter cell culture suspension plate, filled with 10 milliliters of differentiation medium. Then, place the plate on an orbital shaker set to low speed in the incubator. After another two days, centrifuge the cells at 200 times g for three minutes, and remove the supernatant.
To induce embryoid body differentiation into neural progenitor cells, resuspend the cells in differentiation medium with 5-micromolar retinoic acid. Incubate the plate for two days. Then, replace the least half of the medium with fresh medium by tilting the plate and pipetting it out.
