This work was supported by the Neurotrauma Research Program (Western Australia). This project is funded through the Road Trauma Trust Account, but does not reflect views or recommendations of the Road Safety Council.

1. Optical Calibration: Measuring Light Output
<ol>
	<li>To prepare the light delivery apparatus, connect a broadband light source (e.g., Xenon or tungsten lamp) to an appropriate power supply. Position a collimating lens in front of the light source to produce a collimated beam of light. Pass the light through a liquid heat filter to remove most of the heat from the light beam. Depending on the application, focus the collimated beam on to the entrance aperture of a liquid light guide, which provides for more flexible delivery of the light (e.g., into an incubator).</li>
	<li>At the other end of the liquid light guide, position a second collimating lens and then a holder for the interference and neutral density filters. Check that this arrangement produces an evenly illuminated spot of light of the desired waveband and intensity. 
	Note: The distance between the end of the light guide and the specimen plane may need to vary depending on the area that must be illuminated, but remember that as the distance from the end of the light guide increases, light intensity will decrease. In the present study, this distance is 14cm and produces a beam cross section that evenly illuminates a 3x3 well area on a 96-well plate.</li>
	<li>Ensure that stray light from the lamp and associated optical components is unable to reach the specimen.</li>
	<li>Turn on the water cooler for the liquid heat filter and ensure there is exchange of water through the filter jacket. Turn on the lamp power source and wait for at least 5 min for the broadband light source to stabilize. Note: The use of the water cooler is required to prevent over-heating of the device.</li>
	<li>Select a narrowband interference filter (usually described by their center wavelength, peak transmittance and full width at half maximum (FWHM) bandwidth) to generate the desired waveband of illumination. Note: The narrowband interference filters used in the current study were 442 nm, 550 nm, 671 nm and 810 nm.</li>
	<li>Measure the light produced by the lamp at the plane where the specimen is to be positioned during treatment. Measure light using a calibrated irradiance probe (cosine collector) connected to a suitable spectroradiometer, using propriety software according to manufacturer&#8217;s instructions.</li>
	<li>Use neutral density filters to adjust the intensity of light until the desired output is obtained. Note: In the present study, the intensity of light passed by each interference filter is adjusted to give an equal quantal output at each of the four treatment wavebands. It is important to check the calibration regularly, as the lamp output is subject to change. 
	Note: Following the set up of the apparatus, the cells are ready to be illuminated. Figure 1 is a representation of the light delivery apparatus used in the current study.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52221/52221fig1.jpg" />
Figure 1. Image of the light delivery apparatus. Illustrated are the light power source, xenon lamp with housing, collimating lens, water filter, entrance aperture, liquid light guide, second collimating lens, filter holder, treatment frame and matte black card. Note that the narrowband wavelength and intensity filters are not shown.
2. Cell Preparation
<ol>
	<li>The described R/NIR-LT delivery method can be applied to any cell type or in vitro model system; as such the following descriptions of cell culture are general, using well-established techniques to culture pheochromocytoma (PC12), M&#252;ller (rMC1) and primary mixed retinal cells.</li>
	<li>Prior to cell seeding for light treatment and oxidative stress assay, culture immortalized cells in their respective growth media containing appropriate supplements (e.g., FBS, antibiotics) in T75 flasks until 70-80% confluent.</li>
	<li>Detach immortalised cells from T75 flasks, using the method appropriate for the cell type to be tested e.g., trypsin. If necessary, before proceeding with the seeding of cells in 96-well assay plates, coat assay plate wells with 10&#181;g/ml poly-L-lysine for 1h (e.g., for PC12 cells) or 10&#181;g/ml poly-L-lysine for 1h followed by an O/N incubation with 10&#181;g/ml laminin (e.g., for mixed retinal cells).</li>
	<li>Centrifuge cells to collect as appropriate (e.g., 3 min at 405 x g for PC12 cells or 10 min at 218 x g for rMC1 cells), remove the supernatant and resuspend in 8 ml of appropriate cell culture media.</li>
	<li>If mixed retinal cells are to be used, prepare from P0-5 neonatal rat pups by enzymatic digestion (papain) according to established procedures 19</li>
	<li>Count the number of viable cells excluding 0.4% (w/v) trypan blue dye, using a haemocytometer.</li>
	<li>Adjust cell density such that cells will be approximately 70-80% confluent after 24 or 48h in culture. (for PC12 cells the seeding density is 4.0 x 105 viable cells/ml, for rMC1 cells, it is 2.5 x 105 viable cells/ml and for mixed retinal cells it is 8 x 105 viable cells/ml). After adding cells to either clear or black 96-well plates (used for the H2O2 or DCFH-DA assay respectively), in 100 &#181;l of appropriate growth media, allow plate to sit on a flat surface at 37&#176;C, 5% CO2 (or whatever conditions are appropriate for the specific cell type) for at least 24 hr to allow cell adherence.</li>
	<li>Culture cells in growth media in the appropriate 96 well trays until they are 70-80% confluent (24h for PC12 and rMC1 cells, 48h for mixed retinal cells).</li>
</ol>
3. Adding Glutamate Stressor to Cells
<ol>
	<li>Prepare L-glutamic acid monosodium salt hydrate stressor concentrations of 0-10mM in appropriate full growth culture media.</li>
	<li>Remove media from the cells and gently wash cells 3 times with PBS (PC12) or HBSS (rMC1 and mixed retinal cells). After washes, add glutamate-containing media to a total volume of 100 &#181;l/well.</li>
</ol>
4. First Dosages of Light Treatment
<ol>
	<li>Immediately upon addition of glutamate or the stressor of choice, expose cells to light treatment at the desired wavelengths and intensities by placing under the prepared light delivery apparatus. Be sure to alter the quantal output of the light beam using the established combinations of neutral density filters depending on the wavelength being administered. 
	Note: In the current experiments, expose cells to light treatment for 3 min maintain the temperature by resting the 96 well plates on a 37oC heat pad. Treatment time may be varied as required.
	<ol>
		<li>Place a matte black card underneath the cells to prevent light reflecting into adjacent wells in the 96-well tray designated for other treatment parameters.</li>
	</ol>
	</li>
	<li>If treatment is desired for longer lengths of time, add 25mM Hepes buffer to maintain pH of the cell culture media or ideally, place the apparatus and treatment plates within an incubator with CO2 concentration regulated to 5% CO2, or conditions that are optimal for the specific cell type.</li>
	<li>Following completion of the light treatment, place the cells on a level surface and incubate at 37&#176;C and 5% CO2 (or whatever conditions are optimal for the cell type) for 24h. 
	Note: Additional dosages of R/NIR-LT can be administered as described above, as desired.</li>
</ol>
5. Final Dosage of Light Treatment and Detection of ROS
<ol>
	<li>Before performing the final round of light treatment, prepare the reagents to be used for ROS detection. Prepare 50mM citrate buffer (pH6.0), Triton X100, and H2O2 detection reagent working solution (1:2:97 ratio of 10mM H2O2 detection reagent stock solution; 10 U/ml horseradish peroxidase; 50mM sodium citrate (pH 6.0)). Prepare DCFH-DA at a final concentration of 100&#181;M in appropriate media. 
	Note: DCFH-DA reagent for PC12 cells is in RPMI media, DCFH-DA reagent for rMC1 cells is in DMEM media. Note that commercially available detection reagents have differential sensitivities to specific ROS and reagents should be chosen carefully to provide the information desired for an individual application.</li>
	<li>Administer light treatment to the cells, as described in step 4.1-4.1.2. Ensure the cells are being treated with the desired fluences / wavelengths of light.</li>
	<li>Immediately following light treatment, remove the glutamate-containing media and wash twice with appropriate buffer solution (for PC12 cells, PBS is used and for rMC1 cells, HBSS is used). Perform the ROS assays on the cells as follows:
	<ol>
		<li>H2O2 assay: add 45 &#181;l of 50mM citrate buffer (pH 6.0) and 5 &#181;l Triton X100 to each of the wells. Gently shake the cells on an orbital shaker for 30 sec and incubate at 37&#176;C and 5% CO2 for 15 min. Add 50 &#181;l of H2O2 detection working solution and incubate for 30 min at RT. Measure fluorescence using a plate reader with an excitation wavelength of 530nm and an emission wavelength of 480nm.</li>
		<li>DCF assay: add 100 &#181;l of the 100 &#181;M solution of DCFH-DA to each of the wells and incubate for 30min at 37&#176;C and 5% CO2. Remove the media containing 100 &#181;M DCFH-DA and wash with appropriate buffer solution (as described above) twice. Add 90 &#181;l of buffer solution and 10 &#181;l Triton X100 to each of the wells and gently shake on an orbital shaker for 30 sec before 15 min incubation at 37&#176;C. Measure DCF derived fluorescence using a plate reader with an excitation wavelength of 480nm and an emission wavelength of 530nm.</li>
		<li>Express ROS values relative to the protein concentration of cells remaining in the wells, using a colorimetric kit to quantify protein concentration according to manufacturer&#8217;s instructions, with reference to a standard curve to calculate mg protein.</li>
	</ol>
	</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+reactive+oxygen+species.">Mitochondria and reactive oxygen species.</a> Free Radical Biology and Medicine. 47, 333-343 (2009).</li><li>Coyle, J. 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The authors declare that they have no competing financial interests.

We have successfully adapted a precise and calibrated light delivery system to provide a mechanism for study of optimization of R/NIR-LT in vitro. Wavelength and intensity parameters of R/NIR-LT are able to be manipulated accurately and effectively using this system. We established that light treatment of the cells did not lead to cell death, although ROS were not reduced at the wavelengths and dosages delivered, in the cell types tested. The maximum intensities achieved by the current system at 670nm (20.11W/m<...

Reactive oxygen species (ROS) are required for a range of signal transduction pathways and normal reactions of cellular metabolism, including those of neuroprotection 1. However, when endogenous antioxidant mechanism are unable to control the production of ROS, cells may succumb to oxidative stress 2,3. Following injury to the CNS, the associated increases in the presence of ROS and oxidative stress are thought to play a substantial role in the progression of damage 4,5. Despite the extensive number of strategies for attenuating oxidative stress that have been assessed, there are currently no completely effective, clinically relevant anti-oxidant strategies for attenuating ROS production and associated oxidative stress in clinical use following neurotrauma 6. Therefore the attenuation of oxidative stress remains an important goal for therapeutic intervention 7.
Improvements following R/NIR-LT have been reported in a wide range of injuries and diseases including reductions in cardial infarct size, renal and hepatic complications during diabetes, retinal degeneration, CNS injury and stroke 8, perhaps by reducing oxidative stress. With particular regard to CNS injury, preclinical studies of efficacy of 670nm light have shown good effects in models of retinal degeneration 9-11, spinal cord injury 12, neuronal death 13. Clinical trials have been conducted for dry age related macular degeneration and are currently underway for stroke 14, however the outcomes of these trials do not appear promising, perhaps due to a failure to employ effective treatment parameters 15. As such, R/NIR-LT has not been widely adopted as part of normal clinical practice in neurotrauma, despite being an easy to administer, non-invasive and relatively inexpensive treatment. Barriers to clinical translation include lack of a clearly understood mechanism of action and absence of a standardized effective treatment protocol 16,17. Current literature regarding light therapy reveals a plethora of variation in treatment parameters with respect to irradiation sources (LED or laser), wavelength (e.g., 630, 670, 780, 810, 830, 880, 904nm), total dose (joules of irradiation / unit area), duration (exposure time), timing (pre- or post- insult), treatment frequency and mode of delivery (pulse or continuous) 8. The variability in treatment parameters between studies makes comparison difficult and has contributed to skepticism regarding efficacy 16.
Therefore, optimization of R/NIR-LT is clearly required, with cell culture systems able to provide the high-throughput screening mechanism necessary to compare the multiple variables. However there are few commercially available illumination systems that can provide sufficient flexibility and control over wavelength and intensity to perform such optimization experiments. Commercially available LED devices are generally not able to deliver multiple wavelengths or intensities, resulting in investigators employing multiple LED devices from different manufacturers, which may vary not only in the intensity, but also the spectrum of wavelength of light emitted. We have addressed this issue by employing a broadband Xenon light source filtered through narrowband interference filters, thereby generating a range of wavelengths and fluences of light, allowing close, accurate control of the parameters of R/NIR-LT.
It is important to note that the therapeutic dose of treatment is defined by the number of photons interacting with the photoacceptor (chromophore), which, in the case of R/NIR-LT is postulated to be cytochrome c oxidase (COX) 18. Photon energy delivered varies with wavelength; meaning equal doses of energy at different wavelengths will be comprised of different numbers of photons. Therefore, the light emitted from the device was calibrated to emit an equal number of photons for each of the chosen wavelengths to be tested. We have developed a system that can be used to deliver R/NIR-LT at a range of wavelengths and intensities to cells in vitro and demonstrated the ability to measure the effects of the delivered R/NIR-LT on ROS production in cells subjected to glutamate stress.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>OxiSelect Intracellular ROS Assay Kit (Green Fluorescence)</td>
			<td>Cell Biolabs</td>
			<td>STA-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex UltraRed Reagent</td>
			<td>Molecular Probes</td>
			<td>A36006</td>
			<td></td>
		</tr>
		<tr>
			<td>300 Watt Xenon Arc Lamp</td>
			<td>Newport Corporation</td>
			<td>6258</td>
			<td>Very intense light source, do not look directly into the lamp. Ensure there is sufficient cooling to the lamp whilst it is switched on</td>
		</tr>
		<tr>
			<td>USB4000-FL Fluorescence Spectrometer</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>CC-3-UV Cosine Corrector for Emission Collection</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;m diameter quartz fibre optic</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraSuite Spectroscopy Platform</td>
			<td>Ocean Optics</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2300 EnSpire Multimode Plate Reader</td>
			<td>Perkin Elmer</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Acute toxicity, wear gloves when handling.</td>
		</tr>
		<tr>
			<td>L-Glutamic acid monosodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>142-47-2 (anhydrous)</td>
			<td></td>
		</tr>
		<tr>
			<td>Pheochromocytoma rat adrenal medulla (PC12) cells</td>
			<td>American Type Culture Collection</td>
			<td>CRL-2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI1640) Media</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
			<td>Gibco</td>
			<td>10082-147</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Horse Serum, New Zealand origin</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100 mM Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>100X MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Retinal Muller (rMC1) cells</td>
			<td>University of California, San Diego</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>75cm2 Flasks</td>
			<td>BD Biosciences</td>
			<td>B4-BE-353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-134</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Gibco</td>
			<td>23017-015</td>
			<td>Aliquot and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>1X Neurobasal Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td>Warm to 37&#176;C in water bath before use</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>165U Papain</td>
			<td>Worthington</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>W326305</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 96 well plates, clear bottom, black</td>
			<td>Corning</td>
			<td>CLS3603-48EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Costar Clear Polystyrene 96-Well Plates Untreated; Well shape: Round; Sterile.</td>
			<td>Costar</td>
			<td>07-200-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Seesaw Rocker</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Standard lab epuipment</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Density Filter Paper (0.3)</td>
			<td>THORLABS</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>442nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FL441.6-10</td>
			<td></td>
		</tr>
		<tr>
			<td>550nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB550-10</td>
			<td></td>
		</tr>
		<tr>
			<td>670nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB670-10</td>
			<td></td>
		</tr>
		<tr>
			<td>810nm Bandpass Filter</td>
			<td>THORLABS</td>
			<td>FB810-10e</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.1)</td>
			<td>THORLABS</td>
			<td>NE01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.2)</td>
			<td>THORLABS</td>
			<td>NE02B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.3)</td>
			<td>THORLABS</td>
			<td>NE03B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.5)</td>
			<td>THORLABS</td>
			<td>NE05B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (0.6)</td>
			<td>THORLABS</td>
			<td>NE06B</td>
			<td></td>
		</tr>
		<tr>
			<td>Unmounted &#216;25 mm Absorptive Neutral Density Filters (1.0)</td>
			<td>THORLABS</td>
			<td>NE10B</td>
			<td></td>
		</tr>
	</tbody>
</table>

method for the assessment of effects of a range of wavelengths and intensities of red/near-infrared light therapy on oxidative stress in vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

The output of light delivered at a wavelength of 670nm was calibrated using neutral density filters in order to irradiate cells with a range of fluences encompassing a dose of 670nm light previously shown to be beneficial in vivo (0.3 J/cm2) 20. As the number of neutral density filters in front of the light source increased, the intensity (W/m2) decreased, allowing less light to pass to the target area. Table 1 presents the calibration data of 670nm light generated from the light...

Watch this Scientific Journal Video about Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro at JoVE.com

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

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Research

JoVE Journal

Biology

8.6K Views.  The University of Western Australia. Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Video: Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...