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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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/m2) are in excess of previously published measures of penetrance of irradiation into the rat brain, where 1.17 W/m2 reached the ventral surface of the optic nerve and 0.3W/m2 reached the ventral surface of the brain case. When delivered for 30 min, the dose received by cells of the optic nerve in vivo is therefore 0.054 &#8211; 0.31 J/cm2. The doses of 670nm light delivered in the current study (up to 0.38J/cm2) therefore encompass those received by cells in our previous in vivo studies.
The most widely accepted theory of R/NIR-LT efficacy is that of a photoacceptor based system 18, therefore it is imperative to ensure that cells treated with varying wavelengths receive equal quantal fluences of light (photons/cm2/s) 8. In addition, the exposure time used to deliver a particular fluence of light must be kept consistent between treatments, with only the intensity of the light changing to increase and decrease total fluence over time. Previous studies have demonstrated that changing the exposure time to deliver equal fluences results in differences in outcome measures and may act as a confounding factor, and hinder the identification of the optimal wavelength / fluence parameters required for a particular injury model 22,23. As such, we recommend the careful calibration of dosage at each wavelength tested, to ensure useful optimization of R/NIR-LT.
The described technique allows delivery of light to a range of in vitro model systems including organotypic slice cultures, which may result in more meaningful outcomes due to the preservation of cellular architecture and complex inter-cellular interactions. Variations on the injury model may require differences in treatment parameters of R/NIR-LT. This technique is limited by the maximum power output achievable with the described instrumentation. Higher power outputs may be required for some in vitro and most in vivo applications. If higher outputs of light are required, the apparatus may be altered to increase the intensity of light reaching the samples. Shortening the distance between the light termination and target cells will increase the intensity of light reaching the plate. Alternatively, the light may be reflected off a mirror and onto the cells as opposed to being filtered through a liquid light guide, however alternative wavelength and neutral density filters will be required to achieve this. Higher outputs using a more powerful light source would also be required if the method were to be adapted for delivery of R/NIR-LT in in vivo models of CNS injury, due to the increased dosages required to ensure effective penetrance of irradiation 8,20.
In conclusion, the current study describes a novel method of light delivery that provides a means of effectively altering intensity and wavelength parameters of light in R/NIR-LT studies. This methodology will be useful in optimization of R/NIR-LT employing a range of outcome measures and in vitro injury models.

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><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&amp;mu;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 &amp;deg;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 &amp;deg;C in water bath before use</td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050-061</td><td>Warm to 37 &amp;deg;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 &amp;deg;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 &amp;deg;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 &amp;deg;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&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-118</td><td>Warm to 37 &amp;deg;C in water bath before use</td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; 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 &amp;deg;C</td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Gibco</td><td>14025-134</td><td>Warm to 37 &amp;deg;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 &amp;deg;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 &amp;deg;C</td></tr><tr><td>1X Neurobasal Medium</td><td>Gibco</td><td>21103-049</td><td>Warm to 37 &amp;deg;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>442 nm Bandpass Filter</td><td>THORLABS</td><td>FL441.6-10</td><td></td></tr><tr><td>550 nm Bandpass Filter</td><td>THORLABS</td><td>FB550-10</td><td></td></tr><tr><td>670 nm Bandpass Filter</td><td>THORLABS</td><td>FB670-10</td><td></td></tr><tr><td>810 nm Bandpass Filter</td><td>THORLABS</td><td>FB810-10e</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.1)</td><td>THORLABS</td><td>NE01B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.2)</td><td>THORLABS</td><td>NE02B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.3)</td><td>THORLABS</td><td>NE03B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.5)</td><td>THORLABS</td><td>NE05B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;25 mm Absorptive Neutral Density Filters (0.6)</td><td>THORLABS</td><td>NE06B</td><td></td></tr><tr><td>Unmounted &amp;Oslash;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 source fitted with a wavelength filter and includes the number of ND filters used and the intensity of light generated as a result, at described distances from the light output. Fluence, or dose of 670nm light (J/cm2), was calculated from the equation: [Dose (J/cm2) = (Light intensity (W/m2) / 10,000) x time (s)], where the time of treatment was 180s.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Number of ND filters</td>
			<td>Distance from light output (cm)</td>
			<td>Intensity (W/m^2)</td>
			<td>Dose (J/cm^2)</td>
		</tr>
		<tr>
			<td align="right">0</td>
			<td align="right">10.5</td>
			<td align="right">20.11</td>
			<td align="right">0.38</td>
		</tr>
		<tr>
			<td align="right">0</td>
			<td align="right">14</td>
			<td align="right">10.55</td>
			<td align="right">0.19</td>
		</tr>
		<tr>
			<td align="right">1</td>
			<td align="right">14</td>
			<td align="right">4.91</td>
			<td align="right">0.075</td>
		</tr>
		<tr>
			<td align="right">2</td>
			<td align="right">14</td>
			<td align="right">2.28</td>
			<td align="right">0.041</td>
		</tr>
		<tr>
			<td align="right">3</td>
			<td align="right">14</td>
			<td align="right">1.03</td>
			<td align="right">0.018</td>
		</tr>
		<tr>
			<td align="right">4</td>
			<td align="right">14</td>
			<td align="right">0.47</td>
			<td align="right">0.0085</td>
		</tr>
		<tr>
			<td align="right">5</td>
			<td align="right">14</td>
			<td align="right">0.21</td>
			<td align="right">0.0038</td>
		</tr>
		<tr>
			<td align="right">6</td>
			<td align="right">14</td>
			<td align="right">0.094</td>
			<td align="right">0.00169</td>
		</tr>
		<tr>
			<td align="right">7</td>
			<td align="right">14</td>
			<td align="right">0.045</td>
			<td align="right">0.00081</td>
		</tr>
		<tr>
			<td align="right">8</td>
			<td align="right">14</td>
			<td align="right">0.021</td>
			<td align="right">0.000378</td>
		</tr>
		<tr>
			<td align="right">9</td>
			<td align="right">14</td>
			<td align="right">0.013</td>
			<td align="right">0.000234</td>
		</tr>
	</tbody>
</table>
Table 1: Output of the light delivery apparatus fitted with the 670nm wavelength filter.Number of ND filters refers to the number of neutral density filters fitted to the front of the light source output. Intensity (W/m2) refers to the intensity of the light as reported by the propriety software. Fluence, or dose was calculated by the equation[Dose (J/cm2) = (Light intensity (W/m2) / 10,000) x time (s)], where the time of exposure was 180s and distance from the light output was 10.5 or 14 cm.
Two clinically relevant quantal fluences of light were chosen to investigate differential effects of R/NIR-LT wavelength on production of ROS. A dose that can reach CNS tracts following transmission through overlying tissue using LED devices(i.e., 1.78 W/m2 at 670nm) 20 equated to 0.03 J/cm2 for a 3 min&#160;treatment, or 4.9 x 1014 photons/cm2/s. The light source equipped with filters to result in emission of 442, 550, 670 or 830nm was then calibrated using combinations of neutral density filters to emit equal quantal outputs (photons) for each wavelength as opposed to energy outputs (J/cm2), and the dosages (J/cm2) and intensities in W/m2 calculated (Table 2a). An additional higher dose that was within the recommended guidelines to stimulate cellular activity 21 was also used (1.29 x 1015 photons/cm2/s), and calibration conducted for each wavelength (Table 2b).
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Wavelength (&#955;)</td>
			<td>Dose (J/cm^2)</td>
			<td>Intensity (W/m^2)</td>
			<td>Emission (Photons/cm^2/s)</td>
		</tr>
		<tr>
			<td align="right">442</td>
			<td align="right">0.057</td>
			<td align="right">3.21</td>
			<td>4.8 x 10^14</td>
		</tr>
		<tr>
			<td align="right">550</td>
			<td align="right">0.051</td>
			<td align="right">2.87</td>
			<td>5.0 x 10^14</td>
		</tr>
		<tr>
			<td align="right">670</td>
			<td align="right">0.032</td>
			<td align="right">1.78</td>
			<td>4.9 x 10^14</td>
		</tr>
		<tr>
			<td align="right">830</td>
			<td align="right">0.018</td>
			<td align="right">1.01</td>
			<td>4.9 x 10^14</td>
		</tr>
	</tbody>
</table>
Table 2: Calibration of intensity of dosage delivered by equal numbers of photons of light at varying wavelengths. Intensity (W/m2), dosage for a 3 min treatment (J/cm2) and emission (photons/cm2/s) when output of xenon light emitting 442, 550, 670 and 830nm are calibrated to emit A) 4.9 x 1014 photons/cm2/s or B) 1.3 x 1015 photons/cm2/s at a distance of 14 cm from the light output.
In order to assess whether the light delivered by the apparatus was toxic to cells at the dosages used, we assessed the protein content remaining in PC12 cell culture wells following the ROS assays, using a colorimetric protein assay. There was no significant loss of protein at any of the higher output dosages of the light (P &#62; 0.05), indicating that the dosage of light delivered was not causing cell death (Figure 2) and is appropriate to use for assessments of oxidative metabolism.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/52221/52221fig2fixed.jpg" width="500" />
Figure 2: Effect of varying doses of light on the total protein concentration remaining in culture wells following R/NIR-LT and ROS assay. Histogram bars are the mean &#177; S.E.M protein concentrations in PC12 cell culture wells, 6 replicates / concentration, experiments were repeated 3 times. There were no statistically significant differences between control and any of the treatment groups as determined by analysis of variance (ANOVA), p &#62; 0.05.
We initially assessed the effects of 670nm light, delivered at fluences ranging from 0.0085 to 0.38 J/cm2, as an example wavelength to assess suitability of the light delivery apparatus. No significant effect of 670nm light was observed at any of the fluences tested when assessing either H2O2 or DCF fluorescence in PC12, rMC1 or mixed retinal cells stressed with glutamate (Figure 3, P &#62; 0.05). Similarly, there were no significant effects of varying wavelengths of R/NIR-LT delivered at 4.9 x 1014 photons/cm2/s or 1.3 x 1015 photons/cm2/s on ROS production, when assessing H2O2 or DCF fluorescence (P &#62; 0.05, data not shown). Our ability to detect changes in reactive species is confirmed by an increase in fluorescence of the DCFH-DA reactive dye at 13.44 &#177; 0.67 mM glutamate to 22.10 &#177;2.10 at 10mM glutamate. While our data do not reveal positive effects of R/NIR-LT delivered using our light delivery apparatus on ROS production in the selected model system, neither were there negative effects as cells were not compromised, indicated by sustained protein content in culture wells following light therapy (Figure 2). As such, the described method provides a protocol for treating cells or mitochondria with a defined dosage of photons at a range of wavelengths and may be used to assess higher dosages and alternative outcome measures, which may enable optimization of R/NIR-LT parameters.
<img alt="Figure 3" fo:content-width="6.5in" src="/files/ftp_upload/52221/52221fig3fixed.jpg" width="650" />
Figure 3: Quantification of H2O2 (A-C) and DCF (D-F) fluorescence in PC12 (A, D), rMC1 (B, E) or mixed retinal cell (C, F) cultures in the presence of 10mM glutamate stressor, following 670nm irradiation therapy at fluence doses ranging from 0 &#8211; 0.38 J/cm2. Histogram bars represent the mean arbitrary fluorescence units / &#181;g protein &#177; SEM. There were no statistically significant differences between control and any of the treatment groups as determined by ANOVA, p &#62; 0.05, 6 replicates per group, experiments were repeated 3 times.

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 ...

method-for-assessment-effects-range-wavelengths-intensities-rednear

Nanyang Technological University

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JoVE Journal

Biology

8.7K 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

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Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive oxygen species studies. The method utilizes Ortho-phthalaldehyde (OPA) to quantify the cellular concentration of glutathione(s). OPA conjugates with reduced glutathione (GSH) via sulfhydryl binding to subsequently form an isoindole, resulting in a highly fluorescent conjugate. To attain an accurate result of both oxidized glutathione (GSSG) and GSH, a combination of masking agents and reducing agents, which have been implemented in this protocol, are required. Treatments may also impact cellular viability. Hence, normalization via protein assay is presented in this multiparametric assay. The assay demonstrates a pseudo-linear detection range of 0.234 - 30&#181;M (R2=0.9932&#177;0.007 (N=12)) specific to GSH. The proposed assay also allows for the determination of oxidized glutathione with the addition of the masking agent N-ethylmaleimide to bind reduced glutathione, and the reducing agent tris(2-carboxyethyl) phosphine is introduced to cleave the disulfide bond in GSSG to produce two molecules of GSH. The assay is used in combination with a validated bicinchoninic acid assay for protein quantification and an adenylate kinase assay for cytotoxicity assessment.

Author Spotlight: Innovating Thiol Quantification and Biomarker Detection for Oxidative Stress Research 

Quantification of both oxidized and reduced forms of glutathione (GSSG and GSH, respectively) has been achieved through the use of Ortho-phthalaldehyde (OPA). OPA becomes highly fluorescent once conjugated to GSH but is unable to conjugate GSSG until reduced. Here, we describe a multiparametric assay to quantify both using protein quantification for normalization.

Glutathione has long been considered a key biomarker for determining the antioxidant response of the cell. Hence, it is a primary marker for reactive ...

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

Immunology and Infection

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Medicine

Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

Label the glass tubes for the samples to be analyzed, then, add 100 microliters of the prepared sample to each tube. Add 200 microliters of 8.1% SDS to each sample, and gently swirl the glass tube in a circular motion to mix.
Add 1.5 milliliters of 3.5 molar sodium acetate buffer to each sample. Then, add 1.5 milliliters of aqueous 0.8% thiobarbituric acid solution. Bring the final volume in each tube to 4 milliliters by adding 700 microliters of deionized water. Tightly cap the glass tubes, and incubate them in a heating block set to 95 degrees Celsius for 1 hour. Cover the tubes with aluminum foil to prevent condensation. Then, remove the tubes from the block and incubate them on ice for 30 minutes.
After the incubation, centrifuge the samples and standards at 1,500 x g for 10 minutes at 4 degrees Celsius. Transfer 150 microliters of supernatant from each tube to a well in a 96-well plate, removing any air bubbles with a pipette tip.
Measure the absorbance of the samples at 532 nanometers. Then, subtract the average absorbance reading of the blank samples from all other absorbance readings.
Create a standard curve and use it to calculate unknown sample concentrations.

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

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