This work was supported by the Huazhong Scholar Program, the Independent Innovation Fund of the Huazhong University of Science and Technology (No. 2018KFYYXJJ071), and the National Natural Science Foundation of China (Nos. 31501099 and 51707012).

1. Cell Cultures and Predifferentiation
<ol>
	<li>Neural stem cell culture and predifferentiation
	<ol>
		<li>Prepare poly-D-lysine-coated coverslips. Put a sterile coverslip (20 mm in diameter) into a 12-well plate. Coat the cover glass with poly-D-lysine, 0.1% w/v, in water (Table of Materials) for better cell adhesion on the coverslips by following the next steps. 
		NOTE: Optimal conditions must be determined for each cell line and application.
		<ol>
			<li>Aseptically coat the surface of the coverslip with poly-D-lysine, 0.1% w/v, in water. Rock gently to ensure an even coating of the coverslip surface.</li>
			<li>After culturing overnight (~12 h) at 37 &#176;C, remove the poly-D-lysine solution and rinse the surface 3x with 1 mL of sterile water.</li>
			<li>Dry the cells at least 30 min before seeding them.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Murine neural stem cell C17.2 (C17.2-NSC) culture and predifferentiation
	<ol>
		<li>Incubate C17.2-NSCs in a 25 cm2 flask in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 5% horse serum (HS), and 1% penicillin/streptomycin at 37 &#176;C and 5% CO2 for ~2 - 3 d.</li>
		<li>When the cells reach 85% confluence, remove the medium, wash the cells with 1 mL of PBS and add 1 mL of fresh trypsin (0.25%) into the flask. Leave it for 1 min; then, add 1 mL of culture medium to the flask and pipette up and down several times to ensure a single cell suspension.</li>
		<li>Count the density of the cells in the suspension using a hemocytometer. Calculate the required volume of cell suspension to give a final cell concentration of 2 x 104 cells/mL.</li>
		<li>Seed the C17.2-NSCs on the coated coverslips in the 12-well plate with the density of ~2 x 104 cells per well. Check the cell density under a microscope. 
		NOTE: For a better result, the optimal cell density must be determined before the immunofluorescence experiment.</li>
		<li>Incubate the cells at 37 &#176;C in a cell incubator for 12 h to allow for attachment.</li>
		<li>After attachment, wash the cells 2x with 1 mL of PBS and cultivate the cells with differentiation medium consisting of DMEM/F12 with 1% N2 supplement for 48 h before the plasma treatment.</li>
	</ol>
	</li>
	<li>Primary rat neural stem cell culture and predifferentiation
	<ol>
		<li>Culture the primary rat NSC suspension in rat NSC growth medium in uncoated T25 flasks at a density of 5 x 105 cells. 
		NOTE: The primary rat NSCs were isolated following the protocol of Xie et al.9.</li>
		<li>Check the formation of neurospheres under an inverted microscope. Make sure the morphology of the neurospheres exhibits spherical and transparent multicellular complexes.</li>
		<li>When the neurospheres reach a diameter of 3 mm or larger, transfer the neurosphere suspension to a 15 mL sterile centrifuge tube and let the neurospheres settle by gravity.</li>
		<li>Remove the supernatant carefully to leave the neurospheres in a minimal volume of medium.</li>
		<li>Rinse the neurospheres 1x with 5 mL of PBS and let the neurospheres settle by gravity. Remove the supernatant to leave a minimal volume of PBS.</li>
		<li>Add a suitable volume of differentiation medium to adjust the cell density to ~12 - 15 neurospheres per milliliter. The differentiation medium for primary rat NSCs consists of DMEM/F12, 2% B27, and 1% FBS.</li>
		<li>Seed 1 mL of neurosphere suspension onto each coated coverslip in the 12-well plate. 
		NOTE: Shake the plate gently to ensure that the neurospheres are evenly distributed. This step is critical for a better immunofluorescence result.</li>
		<li>Place the plate into the incubator and allow it to predifferentiate for 24 h before the plasma treatment.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Plasma Jets
<ol>
	<li>Choose a half-open quartz tube with an internal diameter of 2 mm and an external diameter of 4 mm. Insert a high-voltage wire with a diameter of 2 mm into the tube.</li>
	<li>Insert the quartz tube with the high-voltage wire into a 5 mL syringe and use a holder to mount it inside the center of the syringe. Set the distance between the sealed end of the quartz tube and the syringe tip at 1 cm.</li>
	<li>Connect a 1 m silicone rubber pipe (with an inner diameter of 12 mm) to the open end of the syringe and, then, connect it to the flowmeter and gas valve in sequence.</li>
</ol>
3. Acquisition of the Jets
<ol>
	<li>Connect the circuit as shown in Figure 1. Connect the output wire of the power supply to the plasma jet device and, then, connect the tip of the high-voltage probe with the output wire to detect the voltage. Connect the other end of the high-voltage probe to the oscilloscope to record the information of the output voltage. Check the whole circuit and make sure the power supply, the oscilloscope, and the high-voltage probe are all grounded.</li>
	<li>Check the gas line. Make sure the gas tube has been connected to the plasma jet device; then, open the gas valve of the helium (volume fraction, 99.999%) and oxygen (volume fraction, 99.999%) and set the gas flow to 1 L:0.01 L/min (He:O2). 
	NOTE: Let the gas flow for several minutes before turning on the power supply for the first time.</li>
	<li>Set the pulse amplitude, frequency, and pulse width as 8 kV, 8 kHz, and 1600 ns, respectively. Check the circuit again and, then, turn on the output button to create a plasma jet. 
	CAUTION: Do not touch the high-voltage wire at any time.</li>
</ol>
4. Plasma Treatment of Neural Stem Cell
<ol>
	<li>Set the distance between the nozzle of the syringe and the cell well hole to 15 mm. 
	NOTE: The distance is measured from the bottom of the platform where the 12-well plate is placed.</li>
	<li>Take the 12-well plate out of the incubator and change the medium of the predifferentiated cells to 800 &#181;L of fresh differentiation medium.</li>
	<li>Divide the cells into three groups: an untreated control group, a 60 s He-and-O2 (1%) gas flow treatment group, and a 60 s plasma treatment group. 
	NOTE: There is no obvious liquid loss under the treatment condition.</li>
	<li>Place the 12-well plate under the plasma jets, make sure the syringe nozzle is fixed in the center of each hole, and give the relevant treatment to the different groups mentioned in step 4.3. 
	NOTE: The untreated controls were kept in differentiation medium at room temperature during the experimental procedure to ensure uniform treatment conditions. The He-and-O2 (1%) gas flow treatment group was treated with only a He and O2 (1%) gas flow, without plasma generation. All the treatments should be performed in triplicate.</li>
</ol>
5. Neural Stem Cell Differentiation
<ol>
	<li>After treatment, remove the original culture and add 1 mL of new differentiation medium to each well.</li>
	<li>Incubate the cells in the incubator at 37 &#176;C and 5% CO2 for 6 d. Change the medium every other day.</li>
	<li>Check the differentiation status of the different groups daily under an inverted phase-contrast light microscope. Randomly select at least 12 fields and take photos to record the morphological changes.</li>
</ol>
6. Immunofluorescence Staining
<ol>
	<li>Rinsing
	<ol>
		<li>Take the samples out of the cell incubator and remove the medium by aspiration. Rinse the cells 1x with 1 mL of PBS. 
		NOTE: After the differentiation, the cells are easily detached. It is necessary to add the PBS from the side of the culture wells to avoid washing off the cells.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Fix the cells with 500 &#181;L of 4% paraformaldehyde for 20 min at room temperature. After the fixation, gently rinse the cells 3x with 1 mL of PBS, 5 min each, to remove the residual 4% paraformaldehyde. 
		NOTE: The optimal time point for cell fixation must be determined according to the cell types. After the fixation, add 1 mL of PBS from the side of the culture wells and leave the sample on the table for 5 min. Do not use a lab shaker, to ensure a minimal loss of cells.</li>
	</ol>
	</li>
	<li>Permeabilization
	<ol>
		<li>Permeabilize the samples with 0.2% TritonX-100 in PBS for 10 min at room temperature. After permeabilization, rinse the cells gently with 1 mL PBS for three times, 5 min each. 
		NOTE: The optimal time point for cell permeabilization must be determined according to cell types. After permeabilization, add 1 mL PBS from the side of the culture wells, leave the sample on the table for 5 min. Do not use a lab shaker to ensure the minimal loss of cells.</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Add 1 mL of 10% goat serum in PBS to each sample and leave the sample on the table for 1 h to block any nonspecific interactions. 
		NOTE: Do not use a lab shaker, to prevent the cells from detaching from the slide. A rinse is not necessary in this step.</li>
	</ol>
	</li>
	<li>Incubation with primary antibody
	<ol>
		<li>Dilute the primary antibody using primary antibody dilution buffer. 
		NOTE: For optimal results, the final dilution ratio of the primary antibody must be determined by pretest. The dilution ratios of anti-Nestin (for undifferentiated stem cell), anti-&#946;-Tubulin III (for neuron), anti-O4 (for oligodendrocyte), anti-NF200 (for mature neurons), anti-ChAT (for cholinergic neurons), anti-LHX3 (for motor neurons), anti-GABA (for GABAergic neurons), anti-serotonin (for serotonergic neurons), and anti-TH (for dopaminergic neurons) are 1:80, 1:200, 1:100, 1:100, 1:100, 1:200, 1:100, 1:200, and 1:100, respectively.</li>
		<li>Apply 300 &#181;L of diluted primary antibodies to different samples.</li>
		<li>Incubate the cell samples overnight at 4 &#176;C. 
		NOTE: It is recommended to incubate primary antibodies at 4 &#176;C to reduce the background and nonspecific staining.</li>
		<li>Remove the primary antibodies and gently rinse the cells 3x with 1 mL of PBS.</li>
	</ol>
	</li>
	<li>Incubation with secondary antibody
	<ol>
		<li>Dilute the Cy3-conjugated or Alexa Fluor 488-conjugated secondary antibodies using 3% goat serum in PBS. 
		NOTE: For optimal results, the final dilution ratio of the secondary antibody must be determined by pretest.</li>
		<li>Apply 300 &#181;L of relevant secondary antibodies to detect each primary antibody. 
		NOTE: All the subsequent steps need to be performed in the dark to prevent fluorescence quenching.</li>
		<li>Incubate the cell sample in the dark for 2 h at room temperature.</li>
		<li>Remove the secondary antibodies and rinse the cells gently with 1 mL of PBS for 10 min at room temperature.</li>
	</ol>
	</li>
	<li>Nuclear staining
	<ol>
		<li>Apply 500 &#181;L of Hoechst 33258 working solution to immerse the cell sample.</li>
		<li>Incubate the cell sample in the dark for 8 min at room temperature to label the nuclei.</li>
		<li>Gently rinse the cells 3x with 1 mL of PBS, 10 min each, protected from light. 
		NOTE: For a minimal loss of cells, the optimal washing condition must be determined empirically.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Place one drop of mounting medium in the center of the microslide.</li>
		<li>Take out of the coverslips (with samples) using tweezers and carefully position the sample on top of the mounting medium. Avoid air bubbles.</li>
		<li>Remove any excess mounting medium with absorbent paper.</li>
	</ol>
	</li>
	<li>Fluorescence microscopy observation
	<ol>
		<li>Observe the samples under the fluorescence microscopy equipped with filters for Hoechst 33258, Alexa Fluor 488, and Cy3. For each sample, randomly select at least 8 - 12 fields and record images with a camera.</li>
	</ol>
	</li>
</ol>

<ol>
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D., von Woedtke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+medicine-current+state+of+research+and+medical+application.">Plasma medicine-current state of research and medical application.</a> Plasma Physics and Controlled Fusion. 59 (1), 014031 (2017).</li><li>Lloyd, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+Plasma:+Medical+Uses+and+Developments+in+Wound+Care.">Gas Plasma: Medical Uses and Developments in Wound Care.</a> Plasma Processes and Polymers. 7 (3-4), 194-211 (2010).</li><li>Yousfi, M., Merbahi, N., Pathak, A., Eichwald, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+plasmas+at+atmospheric+pressure:+toward+new+pharmaceutical+treatments+in+medicine.">Low-temperature plasmas at atmospheric pressure: toward new pharmaceutical treatments in medicine.</a> Fundamental &amp; Clinical Pharmacology. 28 (2), 123-135 (2014).</li><li>Xiong, Z., Roe, J., Grammer, T. 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The authors have nothing to disclose.

C17.2-NSCs is a kind of immortalized neural stem cell line from neonatal mouse cerebellar granular layer cells, developed by Snyder and others10,11. C17.2-NSCs can differentiate into neurons, astrocytes, and oligodendrocytes and are widely used in neuroscience12. In our previous study, CAPs could enhance the differentiation of C17.2-NSCs into neurons. A proof-of-principle study was also carried out using primary rat NSCs, and the effect of...

The directed differentiation of NSCs into a certain lineage for tissue transportation is considered one of the most promising therapies for neurodegenerative and neurotraumatic diseases1. For example, catecholaminergic dopaminergic neurons are especially desired in Parkinson&#39;s disease (PD) treatment. However, traditional methods to prepare the desired cells for transportation have many drawbacks, such as chemical toxicity, scar formation, or others, which largely hampers the applications of NSCs in regenerative medicine2. Therefore, it is very necessary to find a novel and safe way for NSC differentiation.
Plasma is the fourth state of matters, following solid, liquid, and gas, and it constitutes more than 95% of matters in the whole universe. Plasma is electrically neutral with unbound positive/negative and neutral particles and is usually generated by a high-voltage discharge in the lab. In the last two decades, the application of plasma in biomedicine has attracted huge attention worldwide as the development of cold atmospheric pressure plasma technology. Thanks to this technic, stable low-temperature plasma can be generated in the surrounding air at atmosphere without arc formation and consists of various reactive species, such as reactive nitrogen species (RNS), reactive oxygen species (ROS), ultraviolet (UV) radiation, electrons, ions, and electrical field3. CAPs have unique advantages for micro-organism inactivation, cancer therapy, wound healing, treatment of skin diseases, cell proliferation, and cell differentiation4,5,6,7. In previous work, we demonstrated that cold atmospheric plasma jet can enhance the differentiation of NSCs in both murine neural stem cell C17.2 (C17.2-NSCs) and primary rat neural stem cells, exhibiting a great potential to become a powerful tool for the directed differentiation of NSCs8. Although the mechanism of CAP enhancement of NSC differentiation is not fully understood yet, NO generated by CAPs has been proved to be a key factor in the process. In this work, we aim to provide a detailed experimental protocol of using an atmospheric pressure helium/oxygen plasma jet for the treatment of NSCs in vitro, cell preparation and pretreatment, morphology observation by inverted microscope, and fluorescence microscopy observation of immunofluorescence staining.

<table border="0" cellpadding="0" cellspacing="0" style="width:849px;" width="849">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Coverslip</td>
			<td style="width:123px;">NEST</td>
			<td style="width:344px;">801008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Poly-D-lysine</td>
			<td>Beyotime</td>
			<td>P0128</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM medium</td>
			<td>HyClone</td>
			<td>SH30022.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DMEM/F12 medium</td>
			<td>HyClone</td>
			<td>SH30023.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">B27 supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Donor Horse serum</td>
			<td>HyClone</td>
			<td>SH30074.03</td>
			<td>tored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin/Streptomycin</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin</td>
			<td>HyClone</td>
			<td>25300054</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS solution</td>
			<td>HyClone</td>
			<td>SH30256.01B</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0098</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TritonX-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Normal Goat Serum Blocking Solution</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-Nestin</td>
			<td>Beyotime</td>
			<td>AF2215</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-&#946;-Tubulin III</td>
			<td>Sigma Aldrich</td>
			<td>T2200</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">anti-O4</td>
			<td>R&#38;D Systems</td>
			<td>MAB1326</td>
			<td>stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">anti-NF200</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">anti-ChAT</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">anti- LHX3</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti-GABA</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti-Serotonin</td>
			<td style="width:123px;">Abcam, Cambridge, MA</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti-TH</td>
			<td style="width:123px;">Abcam, Cambridge, MA</td>
			<td style="width:344px;"></td>
			<td style="width:167px;">stored at -20 &#176;C, avoid repeated freezing and thawing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunol Staining Primary Antibody Dilution Buffer</td>
			<td>Beyotime</td>
			<td>P0103</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy3 Labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0516</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488- Labeled Goat</td>
			<td>Beyotime</td>
			<td>A0428</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Mouse IgG&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-well plate</td>
			<td>corning</td>
			<td>3512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 cm2 flask</td>
			<td>corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258</td>
			<td>Beyotime</td>
			<td>C1018</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting medium</td>
			<td>Beyotime</td>
			<td>P0128</td>
			<td>stored at -20 &#176;C and protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light microscope</td>
			<td>Nanjing Jiangnan Novel Optics Company</td>
			<td>XD-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microscopy</td>
			<td>Nikon</td>
			<td>80i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High &#8211; voltage Power Amplifier</td>
			<td>Directed Energy</td>
			<td>PVX-4110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DC power supply</td>
			<td>Spellman</td>
			<td>SL1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Function Generator</td>
			<td>Aligent&#160;</td>
			<td>33521A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oscilloscope</td>
			<td style="width:123px;">Tektronix</td>
			<td style="width:344px;">DPO3034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">High voltage probe</td>
			<td style="width:123px;">Tektronix</td>
			<td style="width:344px;">P6015A</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

nerve stem cell differentiation by a one-step cold atmospheric plasma treatment in vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, wound healing, root canal treatment, etc., forming a new research field named plasma medicine. Being a mixture of electrical, chemical, and biological reactive species, CAPs have shown their abilities to enhance nerve stem cells differentiation both in vitro and in vivo and are becoming a promising way for neurological disease treatment in the future. The much more exciting news is that using CAPs may realize one-step, and safely directed, differentiation of neural stem cells (NSCs) for tissue transportation. We demonstrate here the detailed experimental protocol of using a self-made CAP jet device to enhance NSC differentiation in C17.2-NSCs and primary rat neural stem cells, as well as observing the cell fate by inverted and fluorescence microscopy. With the help of immunofluorescence staining technology, we found both the NSCs showed an accelerated differential rate than the untreated group, and ~75% of the NSCs selectively differentiated into neurons, which are mainly mature, cholinergic, and motor neurons.

Cell morphology was observed under the inverted microscope every day after the CAP treatment. Figure 2 shows the ordinary inverted phase-contrast light microscope images of the cell differentiation in both cell lines. The plasma-treated group exhibits an accelerated differentiation rate and a high differentiation ratio compared to the control and gas flow group.
The immunofluorescen...

Watch this Scientific Journal Video about Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro at JoVE.com

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement.

Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

As the development of physical plasma technology, cold atmospheric plasmas (CAPs) have been widely investigated in decontamination, cancer treatment, ...

nerve-stem-cell-differentiation-one-step-cold-atmospheric-plasma

Research

JoVE Journal

Engineering

7.3K Views.  Huazhong University of Science and Technology. This protocol aims to provide detailed experimental steps of a cold atmospheric plasma treatment on neural stem cells and immunofluorescence detection for differentiation enhancement. 

Video: Nerve Stem Cell Differentiation by a One-step Cold Atmospheric Plasma Treatment In Vitro

Investigation of Early Plasma Evolution Induced by Ultrashort Laser Pulses

Early plasma is generated owing to high intensity laser irradiation of target and the subsequent target material ionization. Its dynamics plays a significant role in laser-material interaction, especially in the air environment1-11.
Early plasma evolution has been captured through pump-probe shadowgraphy1-3 and interferometry1,4-7. However, the studied time frames and applied laser parameter ranges are limited. For example, direct examinations of plasma front locations and electron number densities within a delay time of 100 picosecond (ps) with respect to the laser pulse peak are still very few, especially for the ultrashort pulse of a duration around 100 femtosecond (fs) and a low power density around 1014 W/cm2. Early plasma generated under these conditions has only been captured recently with high temporal and spatial resolutions12. The detailed setup strategy and procedures of this high precision measurement will be illustrated in this paper. The rationale of the measurement is optical pump-probe shadowgraphy: one ultrashort laser pulse is split to a pump pulse and a probe pulse, while the delay time between them can be adjusted by changing their beam path lengths. The pump pulse ablates the target and generates the early plasma, and the probe pulse propagates through the plasma region and detects the non-uniformity of electron number density. In addition, animations are generated using the calculated results from the simulation model of Ref. 12 to illustrate the plasma formation and evolution with a very high resolution (0.04 ~ 1 ps).
Both the experimental method and the simulation method can be applied to a broad range of time frames and laser parameters. These methods can be used to examine the early plasma generated not only from metals, but also from semiconductors and insulators.

An experimental method to examine the early plasma evolution induced by ultrashort laser pulses is described. Using this method, high quality images of early plasma are obtained with high temporal and spatial resolutions. A novel integrated atomistic model is used to simulate and explain the mechanisms of early plasma.

Early plasma is generated owing to high intensity laser irradiation of target and the subsequent target material ionization. Its dynamics plays a ...

Simulation of the Planetary Interior Differentiation Processes in the Laboratory

A planetary interior is under high-pressure and high-temperature conditions and it has a layered structure. There are two important processes that led to that layered structure, (1) percolation of liquid metal in a solid silicate matrix by planet differentiation, and (2) inner core crystallization by subsequent planet cooling. We conduct high-pressure and high-temperature experiments to simulate both processes in the laboratory. Formation of percolative planetary core depends on the efficiency of melt percolation, which is controlled by the dihedral (wetting) angle. The percolation simulation includes heating the sample at high pressure to a target temperature at which iron-sulfur alloy is molten while the silicate remains solid, and then determining the true dihedral angle to evaluate the style of liquid migration in a crystalline matrix by 3D visualization. The 3D volume rendering is achieved by slicing the recovered sample with a focused ion beam (FIB) and taking SEM image of each slice with a FIB/SEM crossbeam instrument. The second set of experiments is designed to understand the inner core crystallization and element distribution between the liquid outer core and solid inner core by determining the melting temperature and element partitioning at high pressure. The melting experiments are conducted in the multi-anvil apparatus up to 27 GPa and extended to higher pressure in the diamond-anvil cell with laser-heating. We have developed techniques to recover small heated samples by precision FIB milling and obtain high-resolution images of the laser-heated spot that show melting texture at high pressure. By analyzing the chemical compositions of the coexisting liquid and solid phases, we precisely determine the liquidus curve, providing necessary data to understand the inner core crystallization process.

The high-pressure and high-temperature experiments described here mimic planet interior differentiation processes. The processes are visualized and better understood by high-resolution 3D imaging and quantitative chemical analysis.

A planetary interior is under high-pressure and high-temperature conditions and it has a layered structure. There are two important processes that led to ...

Measurement of Coherence Decay in GaMnAs Using Femtosecond Four-wave Mixing

The application of femtosecond four-wave mixing to the study of fundamental properties of diluted magnetic semiconductors ((s,p)-d hybridization, spin-flip scattering) is described, using experiments on GaMnAs as a prototype III-Mn-V system.&#160; Spectrally-resolved and time-resolved experimental configurations are described, including the use of zero-background autocorrelation techniques for pulse optimization.&#160; The etching process used to prepare GaMnAs samples for four-wave mixing experiments is also highlighted.&#160; The high temporal resolution of this technique, afforded by the use of short (20 fsec) optical pulses, permits the rapid spin-flip scattering process in this system to be studied directly in the time domain, providing new insight into the strong exchange coupling responsible for carrier-mediated ferromagnetism.&#160; We also show that spectral resolution of the four-wave mixing signal allows one to extract clear signatures of (s,p)-d hybridization in this system, unlike linear spectroscopy techniques.&#160;&#160; This increased sensitivity is due to the nonlinearity of the technique, which suppresses defect-related contributions to the optical response. This method may be used to measure the time scale for coherence decay (tied to the fastest scattering processes) in a wide variety of semiconductor systems of interest for next generation electronics and optoelectronics.

The technique of femtosecond four-wave mixing is described, including spectrally-resolved and time-resolved configurations. We illustrate the utility of this technique for the investigation of crucial physical properties in the III-V diluted magnetic semiconductors, afforded by its nonlinearity and high temporal resolution.

The application of femtosecond four-wave mixing to the study of fundamental properties of diluted magnetic semiconductors ((s,p)-d hybridization, ...

How to Ignite an Atmospheric Pressure Microwave Plasma Torch without Any Additional Igniters

This movie shows how an atmospheric pressure plasma torch can be ignited by microwave power with no additional igniters. After ignition of the plasma, a stable and continuous operation of the plasma is possible and the plasma torch can be used for many different applications. On one hand, the hot (3,600 K gas temperature) plasma can be used for chemical processes and on the other hand the cold afterglow (temperatures down to almost RT) can be applied for surface processes. For example chemical syntheses are interesting volume processes. Here the microwave plasma torch can be used for the decomposition of waste gases which are harmful and contribute to the global warming but are needed as etching gases in growing industry sectors like the semiconductor branch. Another application is the dissociation of CO2. Surplus electrical energy from renewable energy sources can be used to dissociate CO2 to CO and O2. The CO can be further processed to gaseous or liquid higher hydrocarbons thereby providing chemical storage of the energy, synthetic fuels or platform chemicals for the chemical industry. Applications of the afterglow of the plasma torch are the treatment of surfaces to increase the adhesion of lacquer, glue or paint, and the sterilization or decontamination of different kind of surfaces. The movie will explain how to ignite the plasma solely by microwave power without any additional igniters, e.g., electric sparks. The microwave plasma torch is based on a combination of two resonators &#8212; a coaxial one which provides the ignition of the plasma and a cylindrical one which guarantees a continuous and stable operation of the plasma after ignition. The plasma can be operated in a long microwave transparent tube for volume processes or shaped by orifices for surface treatment purposes.

This movie shows how an atmospheric plasma torch can be ignited by microwaves with no additional igniters and provides a stable and continuous plasma operation suitable for plenty of applications.

This movie shows how an atmospheric pressure plasma torch can be ignited by microwave power with no additional igniters. After ignition of the plasma, a ...

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-&#946;1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium ...

Applying X-ray Imaging Crystal Spectroscopy for Use as a High Temperature Plasma Diagnostic

X-ray spectra provide a wealth of information on high temperature plasmas; for example electron temperature and density can be inferred from line intensity ratios. By using a Johann spectrometer viewing the plasma, it is possible to construct profiles of plasma parameters such as density, temperature, and velocity with good spatial and time resolution. However, benchmarking atomic code modeling of X-ray spectra obtained from well-diagnosed laboratory plasmas is important to justify use of such spectra to determine plasma parameters when other independent diagnostics are not available. This manuscript presents the operation of the High Resolution X-ray Crystal Imaging Spectrometer with Spatial Resolution (HIREXSR), a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma. In addition, this manuscript covers a laser blow-off system that can introduce such ions to the plasma with precise timing to allow for perturbative studies of transport in the plasma.

X-ray spectra provide a wealth of information on high temperature plasmas. This manuscript presents the operation of a high wavelength resolution spatially imaging X-ray spectrometer used to view hydrogen- and helium-like ions of medium atomic number elements in a tokamak plasma.

X-ray spectra provide a wealth of information on high temperature plasmas; for example electron temperature and density can be inferred from line ...

An Atmospheric Pressure Plasma Setup to Investigate the Reactive Species Formation

Non-thermal atmospheric pressure (&#39;cold&#39;) plasmas have received increased attention in recent years due to their significant biomedical potential. The reactions of cold plasma with the surrounding atmosphere yield a variety of reactive species, which can define its effectiveness. While efficient development of cold plasma therapy requires kinetic models, model benchmarking needs empirical data. Experimental studies of the source of reactive species detected in aqueous solutions exposed to plasma are still scarce. Biomedical plasma is often operated with He or Ar feed gas, and a specific interest lies in investigation of the reactive species generated by plasma with various gas admixtures (O2, N2, air, H2O vapor, etc.) Such investigations are very complex due to difficulties in controlling the ambient atmosphere in contact with the plasma effluent. In this work, we addressed common issues of &#39;high&#39; voltage kHz frequency driven plasma jet experimental studies. A reactor was developed allowing the exclusion of ambient atmosphere from the plasma-liquid system. The system thus comprised the feed gas with admixtures and the components of the liquid sample. This controlled atmosphere allowed the investigation of the source of the reactive oxygen species induced in aqueous solutions by He-water vapor plasma. The use of isotopically labelled water allowed distinguishing between the species originating in the gas phase and those formed in the liquid. The plasma equipment was contained inside a Faraday cage to eliminate possible influence of any external field. The setup is versatile and can aid in further understanding the cold plasma-liquid interactions chemistry.

An experimental setup was created for the helium-operated kHz frequency plasma jet. The setup includes a cage for the plasma power supply and jet and an in-house built reactor to monitor plasma-induced reactive species without the interference of the ambient atmosphere.

Non-thermal atmospheric pressure (&#39;cold&#39;) plasmas have received increased attention in recent years due to their significant biomedical potential. ...

Spark Plasma Sintering Apparatus Used for the Formation of Strontium Titanate Bicrystals

A spark plasma sintering apparatus was used as a novel method for diffusion bonding of two single crystals of strontium titanate to form bicrystals with one twist grain boundary. This apparatus utilizes high uniaxial pressure and a pulsed direct current for rapid consolidation of material. Diffusion bonding of strontium titanate bicrystals without fracture, in a spark plasma sintering apparatus, is possible at high pressures due to the unusual temperature dependent plasticity behavior of strontium titanate. We demonstrate a method for the successful formation of bicrystals at accelerated time scales and lower temperatures in a spark plasma sintering apparatus compared to bicrystals formed by conventional diffusion bonding parameters. Bond quality was verified by scanning electron microscopy. A clean and atomically abrupt interface containing no secondary phases was observed using transmission electron microscopy techniques. Local changes in bonding across the boundary was characterized by simultaneous scanning transmission electron microscopy and spatially resolved electron energy-loss spectroscopy.

A viable technique for the formation of strontium titanate bicrystals at high pressure and fast heating rate via the spark plasma sintering apparatus is developed.

A spark plasma sintering apparatus was used as a novel method for diffusion bonding of two single crystals of strontium titanate to form bicrystals with ...

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.

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 ...

Atmospheric Pressure Fabrication of Large-Sized Single-Layer Rectangular SnSe Flakes

Tin selenide (SnSe) belongs to the family of layered metal chalcogenide materials with a buckled structure like phosphorene, and has shown potential for applications in two-dimensional nanoelectronics devices. Although many methods to synthesize SnSe nanocrystals have been developed, a simple way to fabricate large-sized single-layer SnSe flakes remains a great challenge. Herein, we show the experimental method to directly grow large-sized single-layer rectangular SnSe flakes on commonly used SiO2/Si insulating substrates using a straightforward two-step fabrication method in an atmospheric pressure quartz tube furnace system. The single-layer rectangular SnSe flakes with an average thickness of ~6.8 &#197; and lateral dimensions of about 30 &#181;m &#215; 50 &#181;m were fabricated by a combination of vapor transport deposition technique and nitrogen etching route. We characterized the morphology, microstructure, and electrical properties of the rectangular SnSe flakes and obtained excellent crystallinity and good electronic properties. This article about the two-step fabrication method can help researchers grow other similar two-dimensional, large-sized, single-layer materials using an atmospheric pressure system.

A protocol is presented demonstrating a two-step fabrication technique to grow large-sized single-layer rectangular shaped SnSe flakes on low-cost SiO2/Si dielectrics wafers in an atmospheric pressure quartz tube furnace system.

Tin selenide (SnSe) belongs to the family of layered metal chalcogenide materials with a buckled structure like phosphorene, and has shown potential for ...