Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro
March 21st, 2015
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.
The overall goal of the following procedure is to assess the effects of a range of wavelengths and intensities of red near infrared light therapy on oxidative stress in vitro. In order to do this, we have developed a novel method of delivering the light where we can manipulate the wavelength and intensity delivered. We mount a xenon or broadband light source in a housing on a metal base, then place a coating lens, water filter, and entrance aperture in front of the light.
The next step of the procedure is to guide the light generated from the broadband light source through the entrance, aperture, and liquid light guide and through a second coating lens. Then bandwidth and neutral density filters are fitted to obtain the desired wavelength and intensity of the light. Note that the filters allow passage of a narrow range of wavelengths referred to as a waveband.
The main advantage of this technique over existing methods of red near infrared light therapy delivery, such as LEDs or lasers, is that it allows precise and calibrated manipulation over wavelength and intensity parameters. This then allows us to optimize the delivery of the light for the best biological outcomes the current study administers like to disassociated cells. In addition, it may also be applied to other Systems such as organotypic, models of neurotrauma.
When working with any Bright broadband light source, ensure that you do not look directly into the source of light as it may result in permanent visual damage. To prepare the light delivery apparatus, connect a broadband light source such as a xenon or tungsten lamp to an appropriate power supply. Position a collating lens in front of the light source to produce a collated beam of light.
Pass the light through a liquid heat filter to remove most of the Heat from the light beam. Position a collating lens in front of the light source to produce a collated 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 collated beam to the entrance arture of a liquid light guide to provide more flexible delivery of the light. At the other end of the liquid light guide position a second coating lens and then a holder for the interference and neutral density filters. This arrangement should produce an evenly illuminated spot of light of the desired waveband and intensity.
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. It is important to remember that in accordance with the inverse square law, as the distance from the end of the light guide increases light intensity will decrease in the present study. This distance is 14 centimeters, which results in the production of a beam cross section that evenly illuminates a three by three well area on a 96 well tissue culture plate.
Ensure that stray light from the lamp and associated optical components is unable to reach other specimens, for example, using black cardboard. Once the device is constructed, turn on the water cooler for the liquid heat filter and ensure there is an exchange of water through the filter jacket. Turn on the lamp power source and wait for at least five minutes for the broadband light source.
To Stabilize, select a narrow band interference filter To generate the desired waveband of illumination. Measure the light produced by the lamp at the plane where the specimen is to be positioned. During treatment.
Light is measured using a calibrated irradiance probe connected to a suitable spectra.Radiometer. Use neutral density filters to adjust the intensity of light Until the desired output is obtained. In the present study, the intensity Of light passed by each interference filter is adjusted to give an equal qual output at each of the four Treatment wave bands.
The described red near-infrared Light therapy delivery method can be applied to any cell type or in vitro model system. Here we show our technique of delivery of light to cells using pheochromocytoma or PC 12 cells. As an example, culture cells in growth media.
In 96, well trays coated with polyol lysine until they're 70 to 80%confluent. Prepare the desired concentration Of glutamate or alternative stressor in full growth culture media. Here we use 10 millimolar glutamate.
Remove media from the cells and gently wash them three times with phosphate buffer saline. After washing, add glutamate containing media to a total volume of a hundred Microliters per well. Immediately upon addition of glutamate Or the stressor of choice, expose cells to light treatment at the desired wavelengths and intensities by placing the culture plate under the prepared light delivery apparatus.
Be sure to alter the qual output of the light beam using the established combinations of neutral density filters. Depending on the wavelength being administered. Following completion of the light treatment, place the cells on a level surface and incubate at 37 degrees Celsius in carbon dioxide regulated conditions for 24 hours.
Additional doses of red near infrared light therapy can also be administered. Before performing the final round of light treatment, prepare the reagents to be used for detection of reactive oxygen species as an indicator of oxidative stress. Here we use indicated diets that fluoresce when they react with a range of reactive species, including dilu, fluorescein D acetate, administer light treatment to the cells as described in step four, ensure the cells are being treated with the desired flus and wavelengths of light.
Immediately following light treatment, remove the glutamate containing media and wash twice with PBS. Proceed with the assays utilizing indicator dyes that fluoresce when they react with reactive species according to manufacturer's instructions. Measure the fluorescence generated by the indicator dyes using a plate reader set to the appropriate excitation and emission settings.
Use the color of metric kit to quantify the protein concentration in the wells according to the manufacturer's instructions. Express fluorescence outputs relative to the protein concentration for each individual well, and then analyze results using Statistical software. The development of this technique paves the Way for researchers investigating the effects of multiple wavelengths and intensities of red near infrared light therapy on oxidative stress.
This technique may be used in an array of model systems including disassociated cells Or organotypic cultures.
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Overview
This study investigates the effects of red and near-infrared light therapy on oxidative stress in cultured cells. A novel method for delivering light with adjustable wavelengths and intensities was developed to assess its impact on reactive species production in vitro.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Phototherapy
Background
- Oxidative stress is a critical factor in various biological processes.
- Light therapy has been explored for its potential therapeutic effects.
- Understanding the impact of specific wavelengths can enhance therapeutic applications.
- Reactive species production is a key indicator of oxidative stress.
Purpose of Study
- To evaluate the effects of red/near-infrared light therapy on cultured cells.
- To manipulate light parameters for precise assessment.
- To contribute to the understanding of light therapy's role in oxidative stress.
Methods Used
- Utilization of a xenon light source mounted on a metal base.
- Implementation of coating lenses and water filters for light manipulation.
- Use of entrance apertures and liquid light guides to direct light.
- Application of narrow-band interference and neutral density filters to achieve desired wavelengths and intensities.
Main Results
- No significant effects on reactive species production were observed.
- The tested parameters did not influence oxidative stress levels.
- Further exploration may be needed to identify effective light therapy conditions.
- Results contribute to the understanding of light therapy's limitations.
Conclusions
- Red/near-infrared light therapy, under the tested conditions, did not affect oxidative stress.
- Future studies should explore different parameters for potential effects.
- This research adds to the body of knowledge regarding light therapy applications.
