TITLE: LEAP100 Pin-to-Plate Device Protocol for Treating Cancer Cells
AUTHORS: Almha Gilheany, Sebnem Gunes, Laurence Scally, Patrick J Cullen, Brijesh Tiwari, James Curtin, Andressa Maria Aguiar de Carvalho

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Plasma can be described as the fourth state of matter, and as a neutral ionised gas composed of neutral and charged particles, along with electrons, UV-radiation, radicals and electric fields.  CAP is generated at temperatures below 40 ℃ and at near atmospheric pressures rendering it appropriate for use in biomedical applications such as an anti-cancer tool, that has already shown its cytotoxicity effects, selectivity potential and efficacy as a Glioblastoma (GBM) therapy.  </div><div class = "text-block">How CAP interacts with cells depends on many chemical and physical factors, with CAP activity varying with differing plasma discharges, cell types and culture conditions.  Physical factors such as UV and heat have been shown to have had little effect on DNA integrity.  Chemical factors are reported to have a more substantial effect due to the production of reactive oxygen species during CAP treatment.  Characterising a new device is important as different plasma discharge modes arising from plasma-self adaptation leads to different interactions between cells and plasma.  </div><div class = "text-block">The novel device seen here in Figure 1, is a large Pin-to-Plate non-thermal atmospheric plasma generator.  It consists of 88 pin electrodes which are powered by AC Supply (Leap100, PlasmaLeap Technologies, Dublin, Ireland) and has a resonance frequency of 30 to 125 kHz, a discharge frequency of 50 to 3000 Hz with a power range of 50 to 400 W and a discharge gap of a maximum of 55 mm.  The electrodes/samples work area is encased in a fitted container to minimize the escape of Cold Atmospheric Plasma generated reactive species into the environment. </div><div class = "text-block">The position of pins and a plate electrode acts as a ground, reduces power consumption, increases stability and diffuses the discharge across the entire surface.  The discharge has also been seen to diffuse to a larger area than just the pin tip meaning more interactions between reactive species and cells can occur.</div><div class = "text-block">This protocol describes the steps involved in assessing the cytotoxic effects of a novel large Pin-to-Plate non-thermal atmospheric plasma on mammalian cells. </div><div class = "text-block">PURPOSE:  Step-by-step instructions for the use of the Pin-to-Plate device, non-thermal atmospheric plasma, for the treatment of mammalian cells.</div><div class = "text-block">SCOPE:  To provide a standardized protocol for using the Pin-to-Plate device to generate Cold Atmospheric Plasma to treat mammalian cells for use by any laboratory using the Leap100 system device.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dfyybirap.jpg" /></div></div>

[STEPS]
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Install the Electrodes: Pin Electrodes
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Connect the bottom flat electrode to the ground.  The system will not work correctly without this connection.
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Using the High Voltage (HV) wire, connect the bottom electrode to the blue connector of the HV transformer.
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Place the bottom electrode on a flat surface.
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Using the HV wire connect the TOP electrode to the red connector of the HV transformer.
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HV wires must not come into contact with each other and the HV wire connected to the top electrode must not connect to any conductive material.  The distance between these wires and the conductive material must be more than 70 mm.
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Place the top electrode over the bottom electrode and check that both metal plates of the electrodes are parallel.
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Install the HV Transformer
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Remove the black plug from the top of the transformer and replace it with the orange air ventilation plug.  The black plug is used only in transport.
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Connect the HV cable from the top electrode to the red connector (see picture below) at the HV transformer.
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Connect the HV cable from the bottom electrode to the blue connector.
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Connect the input cable to the power supply unit (output).
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Install Power Supply Unit (PSU)
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Connect the cable from the HV transformer to the “OUTPUT” connector.
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Check the range of input voltage and frequency on the production label and connect the input voltage cable if the power supply line is compatible with the version of PSU.
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Check all connections are correct as described thus far.
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Switch ON the main switch.
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The LCD will display the start screen and the version of software used for approximately 4 seconds, the Main Screen is then shown, the STATUS green LED will then slowly flash.  If the ERROR red LED is ON, check the position of the Emergency Switch.
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It is necessary to run an Initial sequence at each power-up or after an emergency stop or after a strong local plasma discharge.
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If the system status is WAIT INIT, the start initialization sequence can be started by pressing INIT Itb (LCD touch button) and pressing the START button.  The Max time between pressing the INIT Itb and the START button is approximately 8 seconds.  INIT Itb will flash during this time.
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Initialization will take around 3 seconds.  If initialization is successful, the STATUS green LED is ON and the System Status is READY.
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To display the RUN screen press RUN Itb.  Before the first start, set low values for the Main parameters.  For Voltage (about 100-150), Duty (30-75), Frequency Discharge (300-500) and Timer (000).
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Check that all safety rules which are described in the Safety Information section are followed.
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The output of PSU is activated by pressing START Itb (LCD touch button) and pressing the START mechanical button within 8 seconds.
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If the system is running, the blue RUN LED is ON and the Status is Running.
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To deactivate the output of the PSU, press the mechanical red STOP button or press the STOP LCD touch button.  In the case of an emergency, the system can be stopped by pressing the EMERGENCY STOP button.  This button should only be used in an emergency.
?. [Set the Desired Parameters]
Set parameters and variables seen below.
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Each parameter value can be changed using the encoder.
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The encoder is turned left or right to change the active value.
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An active value can be chosen by pressing the touch LCD in the position of value.
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When the value is active, the area is inverted (see Configuration on the picture below).
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To save the value into the internal EEPROM, the encoder is pressed (activating the buzzer).  This is also done after both power off and on.
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When parameters are changed while the system is running, the new value will be used immediately and also when the value has not been saved in EEPROM (the encoder was not pressed).
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The 4 most important parameters used to modify plasma discharge are shown on the RUN screen.  These parameters can be changed on the RUN screen or on the CONF screen.
?. [Tune the Plasma Discharge]
Tune the Plasma Discharge
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If plasma discharge is weak, increase the Voltage parameter to its maximum.  If the discharge is still weak, decrease the voltage to around 70% and increase the Duty parameter by one value and again increase Voltage.
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If the plasma discharge has reached an acceptable level, increase the Frequency Discharge parameter to increase the discharge (Pulses) frequency.
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If the plasma discharge is too strong and there is a strong local discharge, deactivate the output and PSU and wait again for initialization.
?. [Sample Treatment]
Sample Treatment
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Seed cells in accordance with the analysis that is intended after treatment.  For assays using a 96-well seed plate, seed cells in 100 μl of the appropriate media.  Use the 24 central wells of the 96 well-plate as indicated in Figure 6  below to ensure symmetry in plate loading to stabilise the plasma field during treatment.
It was found that there were significant IC50 differences in DMEM used with and without the supplementation of 0.11g/L of sodium pyruvate, suggesting sodium pyruvate had a protective effect on cells.  Media without sodium pyruvate should be used when evaluating the full effect of pin-to-plate discharge.
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Leave cells to adhere overnight at 37 ℃ in a humidified atmosphere at a density of 2 x 103 cells/well or 1 x 104 cells/well for 96 hours incubation time post-treatment and 24 hours incubation time post-treatment, respectively.  If using Petri dishes, leave cells to adhere in the same conditions as the well plates but at a density between 0.2 x 105 and 1.2 x 105 cells/dish for a 35mm dish. For a 60mm dish, seed cells at a density between 2.5 x 106 and 3 x 106 cells/dish.
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Ensure that there is 20 μl of media in each well before treatment.  In some assays, the media may need to be fresh.  In this case, remove all media and add 20 μl of fresh media into each well.
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Place the sample (plate without lid) in the centre of the system, at the air gap between the pin electrode and the ground plate as shown below (Figure 7).
N.B  ENSURE CONTAINMENT BOX IS PLACED FIRMLY ON DEVICE. Cell viability is dependent on the containment of the plasma during treatment.  In the absence of containment, no cytotoxicity will be observed.
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Turn on the device by pressing the main switch.
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Start initialization by pressing the INIT Itb LCD touch button and pressing the START button.  The STATUS LED will stop flashing and turn green indicating that the device is ready to use.
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Press the RUN button on the touch LCD.
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Set the parameters using the encoder as described in the SETTING PARAMETERS section above.  A parameter is changed by touching the LCD screen in the position of that value and then turning the encoder left or right to change the active value.
1000Hz was found to be the optimum frequency in previous studies as this produced the greatest combination of Reactive Nitrogen Species and Reactive Oxygen Species within the plasma.  The device has been used to treat cells for up to 320 seconds and was found to be stable, safe and had a cytotoxic effect on cells at a voltage of 240V.  No significant differences were found between the use of 52 μs or 73 μs duty cycles.
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Treat the samples with the plasma using the parameters that were set.
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Following treatment, remove sample plates from the containment box and add 80 μl of fresh media to the wells and incubate at 37 ℃ using 5% CO2 for 96 hours.  96 hours was the optimum incubation time in this study, however, optimum incubation varies between assays.
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Use dimethyl sulfoxide (DMSO) (20%) or 1mM Hydrogen Peroxide (H2O2) as a positive control.
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Cells are now ready for analysis.