Assessing Atmospheric Pressure Plasma Treatment in Glucose-Deprived Neuroblastoma Cells

Abstract

Source: Yan, X., et. al., New Application of an Atmospheric Pressure Plasma Jet as a Neuro-protective Agent Against Glucose Deprivation-induced Injury of SH-SY5Y Cells. J. Vis. Exp. (2017)

The video demonstrates the neuroprotective activity of low-dose atmospheric pressure plasma treatment in glucose-deprived neuroblastoma cells by modulating oxidative stress and enhancing cell viability.

Protocol

1. APPJ (Atmospheric Pressure Plasma Jet) treatment of SH-SY5Y

1. Adjust the distance between the nozzle of the quartz tube and the platform where the 96-well plate will be placed to 3 cm. Ensure that the beam can touch the surface of the culture medium.
   NOTE: The distance is not measured from the bottom of the plate. It is first adjusted to 3 cm between the nozzle of the quartz tube and the platform used to place the 96-well plate.
2. Before the APPJ (Atmospheric Pressure Plasma Jet) treatment, change the culture medium in each well except the control wells to RPMI 1640 without glucose medium.
3. Place the plate under the APPJ nozzle and ensure that the jets can shoot vertically into each well.
4. Treat cells in separate wells with APPJ for 0 s, 1 s, 2 s, 4 s, 8 s, and 12 s.
   NOTE: APPJ is generated by ionizing helium (Figure 1). The cells injured by glucose deprivation are treated by 4 s and 8 s helium flow to eliminate the effects of helium on cells. All the treatments should be performed in triplicate.

2. Cell Viability Assay

NOTE: Do not change the medium in this step.

1. After the APPJ treatment, incubate the cells for 1 h in a cell incubator.
2. Add 10 µL of Cell Counting Kit-8 (CCK-8) solution to each well.
3. Incubate the cells at 37 °C for 4 h.
   NOTE: The SH-SY5Y cell line is sensitive to glucose deprivation conditions. Cell viability decreases to nearly 50% after 1 h of glucose deprivation, which is the optimum cell viability condition for pharmacodynamics studies. CCK-8 has no cytotoxicity to cells, and cells are incubated with CCK-8 reagent for another 4 h in the glucose deprivation conditions after APPJ treatment to check cell viability. After 8 h of glucose deprivation and APPJ treatment, the protective effect of APPJ was significantly reduced because the long duration of glucose deprivation led to severe damage to the SH-SY5Y cells. There was no evidence of living cells after 24 h of glucose deprivation.
4. Measure the absorbance at 450 nm with a microplate reader.

Results

Figure 1: Typical RNS (Reactive nitrogen species) and ROS (Reactive oxygen species) reactions in APP...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SH-SY5Y cell line	China Center for Type Culture Collection	3111C0001CCC000026
RPMI 1640 medium	Thermo Scientific	21875091	Stored at 4 °C
RPMI 1640 medium no glucose	Thermo Scientific	11879020	Stored at 4 °C
Fetal calf serum	Thermo Scientific	16000044	Stored at -20 °C
96 wells plate	corning	3599
Cell Counting Kit-8 (CCK-8)	Dojindo Laboratories	CK04	Stored at 4 °C
Microplate reader	Tecan	M200 Pro	For measuring the absorbance at 450 nm
High – voltage Power Amplifier	Trek	PD06087	For amplifing the power
Function Signal Generator	MaZe Electronics Science&Technology	AT30120	For providing the specific signal
High – Voltage Probe	Tektronix	P6015A	For detecting high voltage
Digital Oscilloscope	Tektronix	DPO4104B	For displaying the signal