eoe sub articles

EoE Sub Articles

1. In vitro&#160;reactive oxygen species (ROS) imaging of cultured retinal ganglion cell (RGC) neurons
<ol>
	<li>On the day of imaging (typically 6-24 h after cell plating), check retinal ganglion cells under a microscope to validate the growth of RGC axons.</li>
	<li>For live-cell imaging, transfer coverslips from the culture dish to a live cell imaging chamber.</li>
	<li>Set up microscope for imaging. Use an inverted microscope equipped with a differential interference contrast (DIC) objective, OG590 long-pass red filter, and an EM-CCD camera.</li>
	<li>Before imaging, replace the zebrafish cell culture medium&#160;with serum and antibiotics (ZFCM)(+)) with zebrafish cell culture medium&#160;without serum and antibiotics (ZFCM(-)).</li>
	<li>Once cells are positioned with 10x objective, acquire images at 60x magnification using a high NA oil immersion objective. Use an additional 1.5x magnification.</li>
	<li>First, acquire DIC images. Then, image roGFP2-Orp1 using an appropriate filter set. Excite roGFP2-Orp1 with 405/20 and 480/30 nm excitation filters sequentially and acquire images with 535/30 nm emission filter after emission light has passed the dichroic mirror 505DCXR.</li>
	<li>After taking the first set of images, exchange media with media containing different treatment solutions. The media should be changed every 30 minutes of imaging to avoid pH and osmolarity changes.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35-mm culture dish</td>
			<td>Sarstedt</td>
			<td>83-3900</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td>2850-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes (100 x 15 mm)</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26140087</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope</td>
			<td>Nikon</td>
			<td>TE2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>ThermoFisher Scientific</td>
			<td>23017-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium with phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>11-415-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium without phenol red</td>
			<td>Gibco/Fisher Scientific</td>
			<td>21-083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma Aldich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma Aldich</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>Steritop 0.22 &#181;m filter</td>
			<td>Millipore</td>
			<td>S2GPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES&#160;</td>
			<td>Sigma Aldrich</td>
			<td>&#160;H4034</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting hydrogen peroxide levels in cultured neurons through live-cell fluorescence imaging

Source: Terzi, A. et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a>&#160;J. Vis. Exp. (2021).
This video demonstrates a technique to detect real-time hydrogen peroxide levels in retinal ganglion cells using the roGFP2-Orp1 biosensor and live cell imaging techniques. It outlines the steps involved in cell preparation, fluorescence imaging, and the quantification of hydrogen peroxide levels.

Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

1. In vitro&#160;reactive oxygen species (ROS) imaging of cultured retinal ganglion cell (RGC) neurons

	On the day of imaging (typically 6-24 h after ...

Take zebrafish retinal ganglion cells cultured on coated coverslips.
These cells express a genetically encoded biosensor that produces the redox-sensitive green fluorescent protein roGFP2 fused to a hydrogen peroxide-sensitive enzyme, Orp1.&#160;
Transfer the coverslip with media to a live cell imaging chamber and place the chamber on an inverted microscope.
Replace the media with serum-free media to reduce background fluorescence.
Due to low hydrogen peroxide levels, roGFP2 remains in its reduced conformation.
Excite the biosensor sequentially at 405 and 480 nm wavelengths.
Reduced roGFP2 exhibits an excitation peak at 480 nm.
Switch to a hydrogen peroxide-containing media.
Hydrogen peroxide enters the cells and reacts with Orp1, oxidizing roGFP2.
Excite the biosensor again.
Oxidized roGFP2 exhibits a fluorescence increase at 405 nm and a decrease at 480 nm.
Calculate the fluorescence intensity ratio.
An increase in the ratio indicates increased cellular hydrogen peroxide levels.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

33 Views. Source: Terzi, A. et al., ROS Live Cell Imaging During Neuronal Development. J. Vis. Exp. (2021). This video demonstrates a technique to detect real-time hydrogen peroxide levels in retinal ganglion cells using the roGFP2-Orp1 biosensor and live cell imaging techniques. It outlines the steps involved in cell preparation, fluorescence imaging, and the quantification of hydrogen peroxide levels. 

Video: Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging - Concept

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

JoVE Journal

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

Bioengineering

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

Transcript

Detecting Hydrogen Peroxide Levels in Cultured Neurons Through Live-Cell Fluorescence Imaging

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti