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 t...

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414 (6859), 112-117 (2001).</li><li>Rossi, F., Cattaneo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+therapy+for+neurological+diseases:+dreams+and+reality.">Neural stem cell therapy for neurological diseases: dreams and reality.</a> Nature Reviews Neuroscience. 3 (5), 401-409 (2002).</li><li>Graves, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+reactive+oxygen+and+nitrogen+species+in+redox+biology+and+some+implications+for+plasma+applications+to+medicine+and+biology.">The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology.</a> Journal of Physics D: Applied Physics. 45 (26), 263001 (2012).</li><li>Weltmann, K. 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. C., Graves, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+Treatment+of+Onychomycosis.">Plasma Treatment of Onychomycosis.</a> Plasma Processes and Polymers. 13 (6), 588-597 (2016).</li><li>Xiong, Z., 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=Selective+neuronal+differentiation+of+neural+stem+cells+induced+by+nanosecond+microplasma+agitation.">Selective neuronal differentiation of neural stem cells induced by nanosecond microplasma agitation.</a> Stem Cell Research. 12 (2), 387-399 (2014).</li><li>Xie, Z., Zheng, Q., Guo, X., Yi, C., Wu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+Culture+and+Identification+of+Neural+Stem+Cells+in+New-born+Rats.">Isolation, Culture and Identification of Neural Stem Cells in New-born Rats.</a> Journal of Huazhong University of Science and Technology. [Med Sci]. 23 (2), 75-78 (2003).</li><li>Ryder, E. F., Snyder, E. Y., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+multipotent+neural+cell+lines+using+retrovirus+vector-mediated+oncogene+transfer.">Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer.</a> Journal of Neurobiology. 21 (2), 356-375 (1990).</li><li>Snyder, E. Y., Taylor, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cell+engraftment+corrects+lysosomal+storage+throughout+the+MRS+VII+mouse+brain.">Neural progenitor cell engraftment corrects lysosomal storage throughout the MRS VII mouse brain.</a> Nature. 374 (6520), 367-370 (1995).</li><li>Snyder, E. Y., Yoon, C., Flax, J. D., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+precursors+can+differentiate+toward+replacement+of+neurons+undergoing+targeted+apoptotic+degeneration+in+adult+mouse+neocortex.">Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 94 (21), 11663-11668 (1997).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Fixatives+for+Immunostaining.">Cell Fixatives for Immunostaining.</a> Methods in Molecular Biology. 588, 55-61 (2010).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+Cell+Membranes.">Permeabilization of Cell Membranes.</a> Methods in Molecular Biology. 588, 63-66 (2010).</li></ol>

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, ...

This method has the potential to enhance the differentiation of neural stem cells in vitro and to contribute to the treatment of neurological disease such as Parkinson's disease. The main advantages of this technique is that the cold atmospheric plasmas may be applied in one step for the safe directed differentiation of neural stem cells for tissue transplantation. Cold atmospheric plasma can enhance neural stem cells differentiation within a short treatment period.And with need more damage to the cells, providing the desired cells for transplantation. Begin by seeding about five times ten to the fifth murine neural stem cells in a 25 centimeter square flask. Containing five milliliters of complete DMEM for two to three days at 37 degrees celsius and five percent carbon dioxide.When the cells reach 85 percent confluency, wash the culture with one milliliter of PBS. Before treatment, with one milliliter of 25 percent trypsin for one minute. When the cells have detached, stop the reaction with one milliliter of culture medium.And pipette several times to generate a single cell suspension. After counting, dilute the cells to a two times ten to the fourth cells per milliliter concentration. Seed one milliliter of stem cells onto one poly-D-lysine coated cover slip per well in nine wells of a 12 cell culture plate.Check the cell density under a microscope. And return the cells to the incubator for 12 hours. When the cells have attached, wash the cover slips two times with one milliliter of PBS per wash, and treat the cells with differentiation medium for 48 hours.For jet acquisition, first connect the output wire of the power supply to the plasma jet device. And the tip of the high voltage probe with the output wire to detect the voltage. Then connect the other end of the high voltage probe to the oscilloscope to record the information from the output voltage.Check the whole circuit to make sure that the power supply, oscilloscope and high voltage probe are all grounded. And check the gas line to make sure that the gas tube is connected to the plasma jet device. Next, open the helium and oxygen gas valves and set the gas flow to a one liter to 01 liters per minute ratio of helium to oxygen.Check the circuit again and press the output button to create a plasma jet. To plasma treat the neural stem cells, set the distance between the syringe nozzle and the first well of cells to 15 millimeters. Replace the supernatants in the neural stem cell cultures with 800 microliters of fresh differentiation medium.And place the plate under the plasma jets. Making sure that the syringe nozzle is fixed over the center of each well. Create three wells with plasma for 60 seconds per treatment, and three wells with one percent helium and oxygen gas for 60 seconds per treatment.Leaving three wells untreated, as controls. Then replace the supernatants with one milliliter of fresh differentiation medium. And return the cells to the incubator for six days.Check the differentiation status of the different groups daily, under an inverted phase contrast light microscope. After the treatment, allow the cells to fully equilibrate in the condition medium for about one hour before replacing the supernatant with fresh differentiation medium. For immunofluorescence analysis, fix the cultures with 500 microliters of four percent paraformaldehyde per well for 20 minutes at room temperature.Followed by three gentle five minute rinses, with one milliliter of PBS per wash. Next, permeabilize the fixed samples with 2 percent Triton X-100 in PBS for 10 minutes at room temperature. Followed by gentle rinsing as demonstrated.After the last wash, block any non specific binding with one milliliter of 10 percent goat serum in PBS per sample for one hour at room temperature. At the end of the incubation, label the cells in each well with 300 microliters of the primary antibody of interest overnight at four degrees celsius. Followed by three washes in PBS as demonstrated.After the last wash, label the cells with 300 microliters of the appropriate secondary antibodies. Incubate for two hours at room temperature, protected from light. Then rinse the cells gently with one milliliter of PBS per well for ten minutes at room temperature.Image the cells on a fluorescence microscope equipped with the appropriate filters. Plasma treated neural stem cells exhibit an accelerated differentiation rate, and a higher differentiation ratio compared to untreated control, and gas flow treated stem cells. After plasma treatment, Nestin decreases.Beta-Tubulin III significantly increases. And O4 slightly increases, compared to control treated and gas flow treated stem cells. Further, after 60 seconds of plasma treatment, a robust expression of mature neuron, cholinergic neuron, and motor neuron markers is observed.With very few GABAergic and serotonergic neurons, and no dopaminergic neurons detected. While attempting these procedures, it is important to remember, treat the cells with proper plasma dosage, because a long and intense plasma treatment period will induce cell apoptosis or necrosis. Remember to also pre test the cell viability before downstream application.

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

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Engineering

7.2K ビュー.  Huazhong University of Science and Technology. この方法は、インビトロにおける神経幹細胞の分化を高め、パーキンソン病などの神経疾患の治療に寄与する可能性を有する。この技術の主な利点は、組織移植のための神経幹細胞の安全な指向分化のために、冷大気プラズマを1つのステップで適用することができるということです。冷たい大気の血漿は、短い治療期間内に神経幹細胞の分化を高めることができる。...

ビデオ: 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.

stimulating nerve stem cell differentiation using cold atmospheric plasma treatment

This video presents a method for triggering differentiation in neural stem cells by utilizing cold atmospheric plasma. This generates UV radiation and reactive molecules to stimulate nerve cells to produce projections, indicating neuronal cell...

Stimulating Nerve Stem Cell Differentiation Using Cold Atmospheric Plasma Treatment

Making sure that the syringe nozzle is fixed over the center of each well, create three wells with plasma for 60 seconds per treatment, and three wells with 1% helium and oxygen gas for 60 seconds per treatment, leaving three wells untreated as controls. Then, replace the supernatants with one milliliter of fresh differentiation medium, and return the cells to the incubator for six days. Check the differentiation status of the different groups daily under an inverted phase contrast light...

treating surfaces with a cold atmospheric pressure plasma using the cost-jet

Treating Surfaces with a Cold Atmospheric Pressure Plasma using the COST-Jet

This protocol is presented to characterize the setup, handling, and application of the COST-Jet for the treatment of diverse surfaces such as solids and...

a step-by-step guide for seeding and differentiation of mesenchymal stem cell cultures for characterization

A Step-by-Step Guide for Seeding and Differentiation of Mesenchymal Stem Cell Cultures for Characterization  

enhancing stem cell differentiation with a transient dmso treatment

This video demonstrates the method for enhancing stem cell differentiation with transient DMSO treatment, which arrests cells in the G1 phase. Post-treatment, an ectodermal differentiation medium with BMP and TGF-&#946; inhibitors is added to promote neural precursor...

Enhancing Stem Cell Differentiation with a Transient DMSO Treatment

After DMSO pretreatment, aspirate media from cells, and add 2 milliliters of differentiation media to each well. Allow the cells to incubate for three to four days at 37 degrees Celsius in a carbon dioxide incubator, replacing media daily with fresh differentiation...

a step by step protocol for subretinal surgery in rabbits

A Step by Step Protocol for Subretinal Surgery in Rabbits

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and...

single-cell sorting of immunophenotyped mesenchymal stem cells from human exfoliated deciduous teeth

Author Spotlight: Importance of Single Cell Sorting in Isolating Purified Populations of Mesenchymal Stem Cells 

This protocol describes the use of fluorescence-activated cell sorting of human mesenchymal stem cells using the single-cell sorting method. Specifically, the use of single-cell sorting can achieve 99% purity of the immunophenotyped cells from a heterogeneous population when combined with a multiparametric flow cytometry-based...

Fluorescence-Activated Single-Cell Sorting of Human Mesenchymal Stem Cells

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optimized protocol for generating functional pancreatic insulin-secreting cells from human pluripotent stem cells

Author Spotlight: Advancements and Challenges in &amp;#946;-Cells Differentiation from Pluripotent Stem Cells

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to...

Differentiating Human Pluripotent Stem Cells into Pancreatic ?-Cells

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neuronal differentiation from mouse embryonic stem cells in vitro

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological...

in vitro human pluripotent stem cell differentiation into insulin-producing pancreatic cells

In Vitro Human Pluripotent Stem Cell Differentiation into Insulin-Producing Pancreatic Cells

one-step protocol for evaluation of the mode of radiation-induced clonogenic cell death by fluorescence microscopy

One-step Protocol for Evaluation of the Mode of Radiation-induced Clonogenic Cell Death by Fluorescence Microscopy

Research on ionizing radiation (IR)-induced clonogenic cell death is important for understanding the effects of IR on malignant tumors and normal tissues. Here, we describe a one-step assay for assessing the major modes of IR-induced clonogenic cell death based on morphology of 4&#39;,6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei visualized by fluorescence...

in vitro differentiation of human pluripotent stem cells into trophoblastic cells

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

in vitro differentiation model of human normal memory b cells to long-lived plasma cells

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation...

an atmospheric pressure plasma setup to investigate the reactive species formation

An Atmospheric Pressure Plasma Setup to Investigate the Reactive Species Formation

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.

regulating schwann cell growth by nanosecond pulsed electric field for peripheral nerve regeneration in vitro

Author Spotlight: Innovative Use of nsPEF to Boost Peripheral Nerve Regeneration

Here, we present a protocol for applying nanosecond pulse electric field (nsPEF) to stimulate Schwann cells in vitro. The synthesis and secretion ability of relevant factors and cell behavior changes validated the successful stimulation using nsPEF. The study gives a positive view of the peripheral nerve regeneration...

Regulating Schwann Cell Growth by Nanosecond Pulsed Electric Field for Peripheral Nerve Regeneration In Vitro

heterotopic renal autotransplantation in a porcine model: a step-by-step protocol

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney...

preparation and in vitro differentiation of mesenchymal stem cell-encapsulated hydrogels for bone formation

Preparation and In Vitro Differentiation of Mesenchymal Stem Cell-Encapsulated Hydrogels for Bone Formation  

a step-by-step method to detect neutralizing antibodies against aav using a colorimetric cell-based assay

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against...

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 ...

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 ...

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. ...

Ice Generation and the Heat and Mass Transfer Phenomena of Introducing Water to a Cold Bath of Brine

We demonstrate a method for the study of the heat and mass transfer and of the freezing phenomena in a subcooled brine environment. Our experiment showed that, under the proper conditions, ice can be produced when water is introduced to a bath of cold brine. To make ice form, in addition to having the brine and water mix, the rate of heat transfer must bypass that of mass transfer. When water is introduced in the form of tiny droplets to the brine surface, the mode of heat and mass transfer is by diffusion. The buoyancy stops water from mixing with the brine underneath, but as the ice grows thicker, it slows down the rate of heat transfer, making ice more difficult to grow as a result. When water is introduced inside the brine in the form of a flow, a number of factors are found to influence how much ice can form. Brine temperature and concentration, which are the driving forces of heat and mass transfer, respectively, can affect the water-to-ice conversion ratio; lower bath temperatures and brine concentrations encourage more ice to form. The flow rheology, which can directly affect both the heat and mass transfer coefficients, is also a key factor. In addition, the flow rheology changes the area of contact of the flow with the bulk fluid.

Here, we present a protocol to demonstrate the generation of ice when water is introduced to a cold bath of brine, as a secondary refrigerant, at a range of temperatures well below the freezing point of water. It can be used as an alternative way of producing ice for industry.

We demonstrate a method for the study of the heat and mass transfer and of the freezing phenomena in a subcooled brine environment. Our experiment showed ...

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 ...