DOI: 10.3791/58663-v
This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.
This method has the potential to enhance the differentiation of neural stem cells in vitro and to contribute to the treatment of neurological disease such as Parkinson's disease. The main advantages of this technique is that the cold atmospheric plasmas may be applied in one step for the safe directed differentiation of neural stem cells for tissue transplantation. Cold atmospheric plasma can enhance neural stem cells differentiation within a short treatment period.
And with need more damage to the cells, providing the desired cells for transplantation. Begin by seeding about five times ten to the fifth murine neural stem cells in a 25 centimeter square flask. Containing five milliliters of complete DMEM for two to three days at 37 degrees celsius and five percent carbon dioxide.
When the cells reach 85 percent confluency, wash the culture with one milliliter of PBS. Before treatment, with one milliliter of 25 percent trypsin for one minute. When the cells have detached, stop the reaction with one milliliter of culture medium.
And pipette several times to generate a single cell suspension. After counting, dilute the cells to a two times ten to the fourth cells per milliliter concentration. Seed one milliliter of stem cells onto one poly-D-lysine coated cover slip per well in nine wells of a 12 cell culture plate.
Check the cell density under a microscope. And return the cells to the incubator for 12 hours. When the cells have attached, wash the cover slips two times with one milliliter of PBS per wash, and treat the cells with differentiation medium for 48 hours.
For jet acquisition, first connect the output wire of the power supply to the plasma jet device. And the tip of the high voltage probe with the output wire to detect the voltage. Then connect the other end of the high voltage probe to the oscilloscope to record the information from the output voltage.
Check the whole circuit to make sure that the power supply, oscilloscope and high voltage probe are all grounded. And check the gas line to make sure that the gas tube is connected to the plasma jet device. Next, open the helium and oxygen gas valves and set the gas flow to a one liter to 01 liters per minute ratio of helium to oxygen.
Check the circuit again and press the output button to create a plasma jet. To plasma 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 one percent 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 condition medium for about one hour before replacing the supernatant with fresh differentiation medium. For immunofluorescence analysis, fix the cultures with 500 microliters of four percent paraformaldehyde per well for 20 minutes at room temperature.
Followed by three gentle five minute rinses, with one milliliter of PBS per wash. Next, permeabilize the fixed samples with 2 percent Triton X-100 in PBS for 10 minutes at room temperature. Followed by gentle rinsing as demonstrated.
After the last wash, block any non specific binding with one milliliter of 10 percent goat serum in PBS per sample for one hour at room temperature. At the end of the incubation, label the cells in each well with 300 microliters of the primary antibody of interest overnight at four degrees celsius. Followed by three washes in PBS as demonstrated.
After the last wash, label the cells with 300 microliters of the appropriate secondary antibodies. Incubate for two hours at room temperature, protected from light. Then rinse the cells gently with one milliliter of PBS per well for ten minutes at room temperature.
Image the cells on a fluorescence microscope equipped with the appropriate filters. Plasma treated neural stem cells exhibit an accelerated differentiation rate, and a higher differentiation ratio compared to untreated control, and gas flow treated stem cells. After plasma treatment, Nestin decreases.
Beta-Tubulin III significantly increases. And O4 slightly increases, compared to control treated and gas flow treated stem cells. Further, after 60 seconds of plasma treatment, a robust expression of mature neuron, cholinergic neuron, and motor neuron markers is observed.
With very few GABAergic and serotonergic neurons, and no dopaminergic neurons detected. While attempting these procedures, it is important to remember, treat the cells with proper plasma dosage, because a long and intense plasma treatment period will induce cell apoptosis or necrosis. Remember to also pre test the cell viability before downstream application.
