Stimulating Nerve Stem Cell Differentiation Using Cold Atmospheric Plasma Treatment

To plasma-treat treat the neural stem cells, set the distance between the syringe nozzle and the first well of cells to 15 millimeters. Replace the supernatants in the neural stem cell cultures with 800 microliters of fresh differentiation medium, and place the plate under the plasma jets.

Making sure that the syringe nozzle is fixed over the center of each well, create three wells with plasma for 60 seconds per treatment, and three wells with 1% helium and oxygen gas for 60 seconds per treatment, leaving three wells untreated as controls. Then, replace the supernatants with one milliliter of fresh differentiation medium, and return the cells to the incubator for six days. Check the differentiation status of the different groups daily under an inverted phase contrast light microscope.

After the treatment, allow the cells to fully equilibrate in the conditioned medium for about one hour before replacing the supernatant with fresh differentiation medium.