New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells
October 9th, 2017
A protocol for the neuroprotective application of low-dose atmospheric pressure plasma treatment on glucose deprivation-induced SH-SY5Y injuries.
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.
View the full transcript and gain access to thousands of scientific videos
Overview
This article presents a protocol for the neuroprotective application of low-dose atmospheric pressure plasma treatment on glucose deprivation-induced injuries in SH-SY5Y cells. The method aims to address key questions in plasma medicine and offers a relatively low-cost treatment option.
Key Study Components
Area of Science
- Neuroscience
- Plasma Medicine
- Cellular Injury
Background
- Glucose deprivation is a common feature in ischemic brain conditions.
- SH-SY5Y cells are a model for studying neuroprotection.
- Atmospheric pressure plasma jets have potential therapeutic applications.
- Previous methods of plasma treatment may cause excessive injuries.
Purpose of Study
- To provide a detailed in-vitro treatment protocol.
- To explore the neuroprotective effects of helium atmospheric pressure plasma.
- To investigate new medical applications of plasma technology.
Methods Used
- Application of low-dose helium atmospheric pressure plasma jets.
- In-vitro treatment of SH-SY5Y cells under glucose deprivation.
- Assessment of neuroprotective effects.
- Comparison with previous plasma treatment methods.
Main Results
- The low-dose plasma treatment effectively reduces injury in SH-SY5Y cells.
- It prevents excessive plasma-induced injuries compared to traditional methods.
- The technique shows promise for ischemic brain therapies.
- Potential applications extend to other systems, including cardiovascular health.
Conclusions
- Low-dose atmospheric pressure plasma can be a viable neuroprotective treatment.
- This method may lead to advancements in plasma medicine.
- Further research is needed to explore its full therapeutic potential.
Frequently Asked Questions
Executive Industry Relevance
This protocol demonstrates a low-dose atmospheric pressure plasma jet approach to mitigate glucose deprivation-induced injury in a human neuroblastoma-derived cell line, offering a mechanistic de-risking strategy for ischemic brain disease models. The dose-dependent cytoprotective effect supports target validation in neuroprotection pathways and enables predictive confidence in early-stage therapeutic screening. The method provides a reproducible in-vitro system for evaluating plasma-based interventions in central nervous system injury models.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses regarding reactive species-mediated neuroprotection under metabolic stress conditions.
- Operational Value: Enables functional target validation by quantifying cell viability changes in response to plasma treatment parameters.
- Predictive Value: Supports preclinical triage by establishing a dose-response relationship for cytoprotection in glucose-deprived neuronal models.
Screening & Assay Development
- Assay Readiness: Prepares validated SH-SY5Y cell models for standardized evaluation of plasma-based cytoprotective agents.
- Quantitative Output: Utilizes Cell Counting Kit 8 to generate reproducible, dose-dependent viability metrics for compound or treatment comparison.
- Platform Scalability: Defines fixed operational parameters (helium flow rate, voltage, frequency, nozzle distance) to ensure assay reproducibility across runs.
Translational & Preclinical Research
- Disease Relevance: Models glucose deprivation, a key feature of ischemic brain injury, to bridge discovery with pathophysiological relevance.
- Mechanistic De-risking: Isolates the effect of plasma treatment from gas flow variables, confirming that observed effects are due to reactive species rather than mechanical shear.
- Translational Continuity: Provides a protocol adaptable to other cell types and injury models, supporting cross-system validation in cardiovascular or neurodegenerative contexts.
Pipeline & Workflow Integration
The method fits within the early discovery continuum, supporting target validation through mechanistic interrogation of plasma-mediated cytoprotection prior to lead identification efforts.
- Discovery Biology: Tests the hypothesis that low-dose atmospheric pressure plasma reduces glucose deprivation-induced injury via reactive nitrogen and oxygen species modulation.
- Screening: Establishes a standardized, reproducible assay format for evaluating neuroprotective compounds or physical treatments under metabolic stress.
- Analytics: Generates quantitative viability readouts at 450 nm absorbance, enabling statistical comparison across treatment doses and time points.
- Translational Research: Connects to ischemic disease models by replicating a core metabolic insufficiency feature observed in stroke and hypoxic injury.
- Enterprise Reuse: Defines a reusable plasma treatment protocol with fixed parameters that can be adapted to other cell lines or injury types with minimal reoptimization.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by demonstrating that plasma-induced cytoprotection is independent of helium flow rate, isolating reactive species as the active component.
- Operational Value: Ensures reproducibility through standardized cell culture, glucose withdrawal, and plasma treatment timing across multi-well formats.
- Strategic Value: Improves go/no-go decision-making by providing a low-cost, high-reproducibility model for early de-risking of neuroprotective strategies.
- Portfolio Impact: Enables risk-adjusted prioritization of plasma-based interventions by establishing a clear optimal dose (8 seconds/well) for maximal viability rescue.
Implementation Considerations
- Requires expertise in plasma physics and cell culture techniques to safely operate high-voltage plasma jet equipment.
- Depends on stable helium gas supply, high-voltage amplification, and precise waveform control via oscilloscope and signal generator.
- Necessitates standardization of cell seeding density, confluence timing, and glucose-free medium exchange across laboratories.
- Involves adaptation of nozzle-to-plate distance and treatment duration when transferring the protocol to different plasma device configurations.
- Limited to low-dose applications to avoid plasma-induced cytotoxicity, necessitating empirical dose-finding for each new cell model or injury condition.
