TITLE: Di-electric barrier discharge cold atmospheric plasma device protocol 
AUTHORS: Aoibheann Flynn, Eline Manaloto, Zhonglei He, Patrick J Cullen, James Curtin, Sean Behan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Plasma, the fourth state of matter, is an ionized gas that can directly interact with cells and tissues. A non-thermal plasma with weak collisions between electrons and heavy particles that is applied under atmospheric conditions is known as cold atmospheric plasma (CAP) (Conway et al., 2019). CAP generates short and long-lived reactive oxygen species (ROS), reactive nitrogen species (RNS), neutral particles, molecules, electrons an electromagnetic field, weak ultraviolet radiation and weak heating affect (Yan et al., 2018) that has lead to its' application in wound healing, sterilization and cancer treatment (von Woedtke et al., 2020). Di-electric barrier discharge devices are one of the most common CAP devices that generate short but wide plasma between an anode and a cathode using atmospheric air, and can be used to intensively treat a large sample area  (Yan et al., 2016). </div><div class = "text-block">The purpose of this protocol is to outline the steps involved in setting-up a di-electric barrier discharge cold atmospheric plasma (DBD-CAP) device for the generation of reactive oxygen species on cell cultures. This protocol provides procedures for direct treatment with and without post treatment storage and also indirect treatment using DBD-CAP on cell cultures. </div></div>

[STEPS]
?. [DBD-CAP device set up]
Place the sample plate in a sterile polypropylene case.
?. [DBD-CAP device set up]
Place a sterile 10mm Perspex layer on top of the polypropylene case.
?. [DBD-CAP device set up]
Place the negative ground electrode (i.e. the bottom electrode) underneath the polypropylene case.
?. [DBD-CAP device set up]
Place the positive electrode (i.e. the top electrode) on top of the Perspex layer.
?. [DBD-CAP device set up]
Measure the distance between the two electrodes. It should be equal to the height of the container and the Perspex layer.
?. [Generation of CAP]
Apply the plasma treatment to the sample for the required time.
?. [Generation of CAP]
After the desired treatment time is completed, turn off the voltage.
?. [Generation of CAP]
Store the sealed samples at room temperature for 24 hours.
?. [Sample analysis]
Analyse Optical Emission Spectroscopies at 200 and 850nm using the Low Straylight Smart CCD Exemplar LS spectrophotometer and OriginPro8.
?. [Generation of CAP]
Remove the sample from the DBD-CAP device, keeping them sealed inside the polypropylene container and bag.
?. [Generation of CAP]
Follow the appropriate step case depending on the intended treatment and post treatment storage.
?. [Generation of CAP]
?. [Generation of CAP]
Remove the high barrier propylene bag containing the treated samples from the DBD-CAP set-up.
It is vital for user safety to ensure this is carried out in the laminar air flow hood, as escaping reactive oxygen species are a health hazard.
?. [Generation of CAP]
Immediately after CAP treatment, remove any remaining media in the plate and replenish with fresh culture media.For 96 well plates, replenish with 100µl fresh culture media.For 6 well plates, replenish with 2ml fresh culture media.For 35mm petri dish, replenish with 2ml fresh culture media.For 60mm petri dish , replenish with 5ml fresh culture media.For 100m petri dish, replenish with 12ml fresh culture media.
?. [Generation of CAP]
Remove the Perspex layer from the sample container.
?. [Generation of CAP]
Place the sample container in the corner of the reactor, outside the plasma discharging area.
?. [Generation of CAP]
Apply the plasma treatment to the sample for the required time.
?. [Generation of CAP]
Once the desired treatment time is completed, turn off the voltage.
?. [Generation of CAP]
Immediately after CAP treatment, remove any remaining media in the plate and replenish with fresh culture media.For 96 well plates, replenish with 100µl fresh culture media.For 6 well plates, replenish with 2ml fresh culture media.For 35mm petri dish, replenish with 2ml fresh culture media.For 60mm petri dish , replenish with 5ml fresh culture media.For 100mm petri dish, replenish with 12ml fresh culture media.
?. [Generation of CAP]
Set the transformer to 75kVRMS at 50 Hz.
?. [Generation of CAP]
Set the transformer to 75kVRMS at 50 Hz.
?. [Generation of CAP]
Set the transformer to 75kVRMS at 50 Hz.
?. [Generation of CAP]
Store the sealed samples in a humidified incubator under 5% (v/v) C02 at 37°C in for 24-48 hours post-treatment storage time.
?. [Generation of CAP]
Apply the plasma to the to the voltage for the required time.
?. [Generation of CAP]
Once the desired treatment time is completed, turn off the voltage.
?. [Generation of CAP]
Remove the sample container containing the samples.
It is vital for user safety to ensure this is carried out in the laminar air flow hood, as escaping reactive oxygen species are a health hazard.
?. [Generation of CAP]
Remove the samples from the container.
It is vital for user safety to ensure this is carried out in the laminar air flow hood, as reactive oxygen species are a health hazard.
?. [Sample preparation]
Plate cells into desired plate (96 well plate, 6 well plate or petri dish (35, 60 or 100mm)) and allow to adhere overnight.
?. [Analysis]
One hour post treatment, measure the fluorescence intensity on a micro-plate reader at 492-495/517-527 nm.
?. [Analysis]
Determination of ROS generation.
?. [Sample preparation]
Remove culture media from the sample, leaving a small amount to avoid drying cells.For 96 well plate, remove culture media to a minimum of 20µl .For 6 well plate, remove culture media to a minimum of 200µl.For a 35mm petri dish, remove culture media to a minimum of 200µl.For a 60mm petri dish, remove culture media to a minimum of 500µl.For a 100mm petri dish, remove culture media to a minimum of 1.2ml.
?. [Generation of CAP]
Store the sealed samples in a humidified incubator under 5% (v/v) C02 at 37°C in for 24-48 hours post-treatment storage time.
?. [Anlysis]
Determination of ROS generation.
?. [Anlysis]
One hour post treatment, measure the fluorescence intensity on a micro-plate reader at 492-495/517-527 nm.
?. [Analysis]
Determination of ROS generation.
?. [Analysis]
One hour post treatment, measure the fluorescence intensity on a micro-plate reader at 492-495/517-527 nm.
?. [DBD-CAP device set up]
The set-up of the DBD-CAP device should resemble the following set-up.
Ensure the electrodes and sample are level and flush to avoid unstable plasma that will arc.
?. [Generation of CAP]
Note the relative humidity and temperature of the atmospheric air.
?. [Generation of CAP]
Note the relative humidity and temperature of the atmospheric air.
?. [Generation of CAP]
Note the relative humidity and temperature of the atmospheric air.
?. [Generation of CAP]
Immediately after CAP treatment, remove any remaining media in the plate and replenish with fresh culture media.For 96 well plates, replenish with 100µl fresh culture media.For 6 well plates, replenish with 2ml fresh culture media.For 35mm petri dish, replenish with 2ml fresh culture media.For 60mm petri dish , replenish with 5ml fresh culture media.For 100m petri dish, replenish with 12ml fresh culture media.
?. [Generation of CAP]
Remove the sample from the DBD-CAP set-up.
It is vital for user safety to ensure this is carried out in the laminar air flow hood, as escaping reactive oxygen species are a health hazard.
?. [Analysis]
Determination of cell viability.
?. [Analysis]
48 hours post treatment, wash cells once with sterile phosphate buffered saline.
?. [Analysis]
Incubate cells for 3 hours at 37°C with 10% alamar blue and 90% culture media solution without fetal bovine serum.
?. [Analysis]
Using a multi-plate reader, measure fluorescence using an excitation wavelength of 520nm and an emission wavelength of 595nm.
?. [Anlysis]
Determination of cell viability.
?. [Anlysis]
48 hours post treatment, wash cells once with sterile phosphate buffered saline.
?. [Anlysis]
Incubate cells for 3 hours at 37°C with 10% alamar blue and 90% culture media solution without fetal bovine serum.
?. [Anlysis]
Using a multi-plate reader, measure fluorescence using an excitation wavelength of 520nm and an emission wavelength of 595nm.