The overall goal of this treatment method is to provide a detailed in-vitro treatment protocol for neuro-protective applications of helium atmospheric pressure plasma jets on glucose deprivation-induced injury in SH-SY5Y cells. This method can help the key questions in the plasma application field such as plasma medicine. The main advantage of this technique is it acts as a plasma treatment relatively low compared to previous methods for consistent treatments.
And we each can prevent excessive plasma-induced injuries. The implications of this technique extend towards therapy of ischemic brain cases because insufficient glucose supply is a common feature of this disease. Though this method can provide insight into new medical applications of plasma it can also be used in other systems such as cardiovascular system.
Generally individuals new to this method may struggle because the treatment parameters must be readjusted for different plasma devices. First connect a high voltage power amplifier to a function signal generator that serves as a power supply to provide an AC signal. To record the waveforms of the the applied voltage to a high voltage electrode, connect one end of the high voltage probe to a digital oscilloscope and connect the other end to the power supply.
Continually pass helium over the quartz tube of the atmospheric pressure plasma jet device and control the gas flow rate at a stable 1.4 standard liter per minute. Next, turn on the oscilloscope, signal generator and high voltage power amplifier. Rotate the frequency adjustment knob to five kilohertz.
Then gradually increase the voltage to a peak to peak value of six kilovolts. After growing an SH-SY5Y human neuroblastoma-derived cell line, carefully aspirate the cell media when the cells reach 85%confluence. Add one milliliter of 0.25%trypsin containing 0.1%EDTA to the cells.
After incubating for 15 seconds at room temperature carefully aspirate the trypsin and add two milliliters of RPMI 1640 containing 15%FBS to neutralize. Gently pipette up and down washing the bottom of each well until the SH-SY5Y monolayer is completely detached. Following this, count the cells with a hemocytometer and adjust the cell concentration by adding RPMI 1640 medium containing 15%FBS.
Then transfer one hundred microliters of cell suspension to each well of a 96 well plate. Adjust the distance between the nozzle of the quartz tube and the platform where the 96 well plate will be placed to three centimeters to ensure that the beam can touch the culture medium surface. Before treatment, change the culture medium in each well to RPMI 1640 without glucose medium.
Now, place the plate under the atmospheric pressure plasma jet nozzle. Then, treat cells in separate wells for zero, one, two, four, eight, and 12 seconds. After treatment, incubate the cells for one hour in a cell incubator at 37 degrees Celsius.
Following incubation, add ten microliters of cell counting kit eight solution to each well. Then incubate the cells at 37 degrees Celsius for four hours. Finally measure the absorbance at 450 nanometers with a microplate reader.
Glucose deprivation reduced the viability of SH-SY5Y cells to 44.1 plus or minus 2.6 percent compared to that of the control group. The atmospheric pressure plasma jet treatment significantly increased cell viability in a dose-dependent manner at an optimal dose of eight seconds per well. And the cell viability reached 62.27 plus or minus 3.1%Gas flow had no effect on the glucose deprivation-induced cell impairment.
Once mastered this technique can be done in 30 hours if it's performed properly. While attempting this procedure it's important to remember to consult all the material safety data sheets before use. After its development, this technology plows a way for researchers in the field of plasma medicine to explore new applications on other kinds of brain injuries and create newer injury models.
After watching this video, you should have a good understanding of how to use an atmospheric pressure plasma jet for new practical applications. Don't forget that working with a high voltage power amplifier can be extremely hazardous and precautions such as not touching the high voltage lines should always be taken when performing this procedure.
