This work was supported by the Innovation fund of Beijing Neurosurgical Institute (2014-11), National Natural Science Foundation of China (Nos. 11475019 and 81271286) and Beijing Natural Science Foundation (No. 7152027).

1. Preparation of the APPJ device
CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate safety practices when performing all the&#160;experiments, including the use of a fume hood and personal protective equipment (safety glasses, protective gloves, lab coat, etc.). The protocol requires standard cell handling techniques (sterilizing, cell recovery, cell passaging, cell freezing, cell staining, etc.).
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No conflicts of interest were declared in relation to this paper.

SH-SY5Y cells are a human neuroblastoma-derived cell line and are widely used as an appropriate cell model for in vitro studies on neurotoxicity or neuroprotection12. The SH-SY5Y cell line was sensitive to glucose deprivation conditions. Cell viability decreased to nearly 50% after 1 h glucose deprivation, which is the optimal cell viability condition for studies of pharmacodynamics. Furthermore, CCK-8 reagent has no cytotoxicity to cells and cells were incubated with CCK-8 reagent for an...

The adult brain almost exclusively uses glucose as a substrate for energy metabolism under normal physiological conditions. The human brain constitutes only 2% of the body weight but consumes approximately 25% of the total glucose within the body1. It is well documented that glucose metabolism dysfunction is one of the major pathological changes during ischemic stroke and various neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Huntington&#39;s disease (HD), and Parkinson&#39;s disease (PD)2,3. The lack of glucose and either impaired glucose uptake or oxidative phosphorylation can directly impact ATP production and further induce neural cell death, which may increase the risk of neuronal dysfunction, suggesting that maintaining cell viability or delaying cell injury after glucose deprivation might be a reasonable approach for treating these diseases. The investigation of neuroprotective effects via glucose modulation, focusing on anti-inflammatory agents, ion channel modulators, free radical scavengers, neurotrophic factors, etc. has been of interest. However, translation of these neuroprotective approaches from bench to clinical practice has not been successful4.
Atmospheric pressure plasma jets (APPJs) are a new kind of atmospheric low temperature gas discharge technology that has attracted the attention of many researchers from multiple disciplines in recent years. APPJs have been used for decades in various biomedical applications such as cancer cell treatment, bacterial inactivation, blood coagulation, wound healing, oral medicine, etc.5,6, due to its emissions of multiple types of reactive nitrogen species (RNS) and reactive oxygen species (ROS) (Figure 1)7. Previous plasma bio-medicine applications mainly focused on the oxidative and/or nitrative stress on bacteria, cells, and tissues8. However, the APPJ could also be a &#34;double-edged sword&#34; since RNS and ROS are important intracellular signaling molecules related to many physiological and pathophysiological processes9. Nitrous oxide (NO) controls a wide range of biological processes and plays a dual role in the human body, especially in central nervous system (CNS). Low levels of NO have shown their neuroprotective activities both in vitro and in vivo via multiple signal pathways10. Our previous study first reported that helium APPJ-induced NO production was involved in the neuroprotective effect of APPJ against oxidative stress-induced injuries11. However, the effects of APPJs on other injuries have not been reported. Therefore, the aim of the present study is to provide an in vitro treatment protocol regarding the neuroprotective applications of helium APPJ on glucose deprivation-induced injury in SH-SY5Y cells. Different from previous studies, our protocol used low-dose plasma treatment for neuroprotective applications without the consequences of excessive plasma-induced injuries, indicating that the APPJ treatment could be potentially used as a novel &#34;NO donor drug&#34; for future research and even for clinical translation. This protocol was also suggested to be used as a cytoprotective application for other cell types with different impairments, but the APPJ treatment conditions should be re-adjusted and the treatment dose must be relatively low.

<table width="911">
	<tbody>
		<tr height="20">
			<td height="20" style="width:216px;height:20px;">SH-SY5Y cell line</td>
			<td style="width:325px;">China Center for Type Culture Collection</td>
			<td style="width:135px;">3111C0001CCC000026</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">RPMI 1640 medium</td>
			<td style="width:325px;">Thermo Scientific&#160;</td>
			<td style="width:135px;">21875091</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">RPMI 1640 medium no glucose</td>
			<td style="width:325px;">Thermo Scientific</td>
			<td style="width:135px;">11879020</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">fetal calf serum</td>
			<td style="width:325px;">Thermo Scientific</td>
			<td style="width:135px;">16000044</td>
			<td style="width:235px;">stored at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">tripsin-EDTA solution</td>
			<td style="width:325px;">Solarbio</td>
			<td style="width:135px;">T1300</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">96 wells plate</td>
			<td style="width:325px;">corning</td>
			<td style="width:135px;">3599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Cell Counting Kit-8 (CCK-8)</td>
			<td style="width:325px;">Dojindo Laboratories</td>
			<td style="width:135px;">CK04</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">microplate reader</td>
			<td style="width:325px;">Tecan</td>
			<td style="width:135px;">M200 Pro</td>
			<td>for measuring the absorbance at 450 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">High &#8211; voltage Power Amplifier</td>
			<td style="width:325px;">Trek</td>
			<td style="width:135px;">PD06087</td>
			<td>for amplifing the power</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Function Signal Generator</td>
			<td style="width:325px;">MaZe Electronics Science&#38;Technology</td>
			<td style="width:135px;">AT30120</td>
			<td>for providing the specific signal</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">High &#8211; Voltage Probe</td>
			<td style="width:325px;">Tektronix</td>
			<td style="width:135px;">P6015A</td>
			<td>for detecting high voltage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">Digital Oscilloscope</td>
			<td style="width:325px;">Tektronix</td>
			<td style="width:135px;">DPO4104B</td>
			<td>for displaying the signal</td>
		</tr>
	</tbody>
</table>

new application of an atmospheric pressure plasma jet as a neuro-protective agent against glucose deprivation-induced injury of sh-sy5y cells

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions include multiple types of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Our previous study has shown the cytoprotective effect of the APPJ against oxidative stress-induced injuries. The aim of the present study is to provide a detailed in vitro treatment protocol regarding the neuroprotective applications of helium APPJs on glucose deprivation-induced injury in SH-SY5Y cells. The SH-SY5Y human neuroblastoma-derived cell line was maintained in RPMI 1640 medium supplemented with 15% fetal calf serum. The culture medium was then changed to RPMI 1640 without glucose before APPJ treatment. After a 1 h incubation in a cell incubator, cell viability was determined using Cell Counting Kit 8. The results showed that, compared to the glucose deprivation group, cells treated with APPJ exhibited significantly increased cell viability in a dose-dependent manner, with 8 s/well observed as an optimal dose. Meanwhile, helium flow had no effect on the glucose deprivation-induced cell impairment. Our results indicated that APPJ could be potentially used as a treatment method for the diseases in the central nervous system related to glucose deprivation. This protocol could also be used as a cytoprotective application for other cells with different impairments, but the cell culture and APPJ treatment conditions should be readjusted, and the treatment dose must be relatively low.

Data are expressed as the mean &#177; SD of at least three independent experiments. Group results were analyzed for variance using ANOVA. All analyses were performed using statistical analysis software Prism and p&#60;0.05 was the threshold for statistical significance.
Cell viability was measured after 4 h of CCK-8 incubation. As shown in Figure 3, glucose deprivation reduced the viability of SH-SY5Y cells to 44.1 &#177; 2.6% compared to that of the control group...

Watch this Scientific Journal Video about New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells at JoVE.com

New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells

A protocol for the neuroprotective application of low-dose atmospheric pressure plasma treatment on glucose deprivation-induced SH-SY5Y injuries.

The atmospheric pressure plasma jet (APPJ) has attracted the attention of many researchers from multiple disciplines in recent years because its emissions ...

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.

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8.9K Views.  Capital Medical University. 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 ex...