Our research program involving the biological effects of ALAN on birds has received funding from the FWO Flanders (to M.E. and R.P., project ID: G.0A36.15N), the University of Antwerp and the European Commission (to M.L.G, Marie Sk&#322;odowska-Curie fellowship ID: 799667). We acknowledge the intellectual and technical support of members of the Behavioral Ecology and Ecophysiology Research group at the University of Antwerp, especially Peter Scheys and Thomas Raap.

All applications of this system to animal experiments were approved by the University of Antwerp&#39;s ethical committee and conducted in accordance with Belgian and Flemish laws. Methodology adhered to the ASAB/ABS guidelines for the use of animals in behavioral research. The Belgian Royal Institute for Natural Sciences (Koninklijk Belgisch Instituut voor Natuurwetenschappen; KBIN) provided licenses for all researchers and personnel.
1. Creating the experimental system
<ol>
	<li>Obtain broad-spectrum LED(s) to use in creating ALAN. Take LED light(s) from a LED headlight. Use either a single LED light or multiple (e.g., 4) broad-spectrum LED lights for more diffuse lighting (Figure 1). 
	NOTE: As a modification, LEDs with different spectral properties (e.g., red versus blue) could be used but would have to be obtained from a different source (see the Supplementary material of Grunst et al. 201915 for the spectral properties of the LEDs used in past studies using this system).</li>
	<li>Design a system to mount the LEDs along with an IR camera to allow for behavioral monitoring. Researchers can accomplish this end in a number of ways.
	<ol>
		<li>Option 1. Insert a single broad-spectrum LED into the nest box separately in a plastic tube adjacent to an IR camera mounted with adhesive on a plastic or metal plate that fits within the nest box (Figure 1A, B).</li>
		<li>Option 2. Mount an IR camera in a central position on a plastic or metal plate and then mount LED lights in fixed positions on the plate surrounding the IR camera (Fig. 1C).</li>
	</ol>
	</li>
	<li>Design a means to connect the system to a power source (battery) and timer.
	<ol>
		<li>Use a knife or drill to make groves in the side of the nest box through which wire connectors can extend to connect the system to a Fe-battery (12 V; 120 Wh) and homemade timer (12 V).</li>
		<li>Design a dark green wooden enclosure that matches the nest box in coloration, length, and width (e.g., nest boxes used in past studies had the dimensions: 120 mm x 155 mm x 250 mm ), and with one side opening via a hinge to house the battery, recorder for the video, and timer system for the LEDs (Figure 2; Supplementary Figure 1 and Supplementary Figure 2).</li>
	</ol>
	</li>
	<li>Design a means through which to adjust ALAN intensity.
	<ol>
		<li>Obtain a resistor (value contingent on battery voltage and illumination) and connect it in series with the LED(s).</li>
	</ol>
	</li>
	<li>Design &#34;dummy&#34; boxes with the same dimensions as the enclosures that house the timer and battery for use in habituating birds to the system (i.e., as in Figure 2A, but without the internal electronics). 
	NOTE: Section 2 and section 3 discuss the step-by-step methods used to study the effects of ALAN on the focal organism.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63381/63381fig01.jpg" /> 
Figure 1: Two systems consisting of IR cameras and LED light(s) used to manipulate ALAN inside nest boxes. (A) Top view of the nest box with plate holding the older system in place. (B) Older system with 1 broad-spectrum LED to manipulate ALAN and central camera with 10 IR LEDs (c) Newer system with 4 broad-spectrum LEDs and central IR camera with 4 IR LEDs. <a href="https://www.jove.com/files/ftp_upload/63381/63381fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63381/63381fig02.jpg" /> 
Figure 2: The homemade battery and timer unit used to manipulate ALAN and video-record behavior. (A) The unit is enclosed within a wooden box that is mounted on top of the nest box. (B) View of the electronics inside the unit. Connectors extend from inside the nest box up into the wooden enclosure to connect the electronics to the IR camera and broad-spectrum LEDs. <a href="https://www.jove.com/files/ftp_upload/63381/63381fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Planning the experiment and adjusting ALAN intensity and timing
<ol>
	<li>Determine the desired light intensity to which to expose animals.
	<ol>
		<li>Carefully consider which experimental light intensity to use so as to produce meaningful results that answer the research question. In general, this will mean selecting an ecologically relevant light intensity, which free-ranging animals are likely to encounter (see Table 1 for guidance).</li>
	</ol>
	</li>
	<li>Adjust the LED lights to the desired light intensity (e.g., 1-3 lux, as used in past studies; Table 1 and Table 2).
	<ol>
		<li>Prior to placement in the field, place the system on a nest box taken into the laboratory to calibrate the light intensity. Connect the LEDs to the power source, as described further below (Protocol section 3).</li>
		<li>Adjust the light emitted by the LEDs to the desired intensity (lux) by placing a light meter at the level of the bird within the nest box (~8 cm from the bottom) and simultaneously adjusting the resistor in series with the LEDs. 
		NOTE: It is possible to achieve very low light intensities (e.g., rural sky glow levels; 0.01 lux).</li>
	</ol>
	</li>
	<li>Determine the timeframe over which to expose animals to ALAN.
	<ol>
		<li>Determine the length and timing of exposure across the night. For instance, one can expose animals to ALAN across the entire night, for only part of the night, or leave a period of darkness in the middle of the night to reduce the degree of perturbation.</li>
		<li>In cases that an animal must enter the nest box (or a specific area) to be exposed to the ALAN, also consider whether the light should be turned on before or after the entry event is likely to occur.</li>
	</ol>
	</li>
	<li>Set the timer to control the period of light exposure during the night.
	<ol>
		<li>Set the timer connected to the broad spectrum LEDs so that the light turns on and off at specified periods (e.g., on at least 2 h before sunset; off 2 h after sunrise). 
		NOTE: The IR camera allows the behavior of the animal to be recorded simultaneously for the duration of the light exposure and will be on as long as it is connected to a charged battery.</li>
	</ol>
	</li>
	<li>Determine the appropriate experimental design to use for the target research question(s). 
	NOTE: For some questions, a repeated measures experimental design will be the most powerful option (e.g., How does exposure to ALAN affect sleep behavior?). For others, paired control and experimental groups will be needed (e.g., How does exposure to ALAN affect telomere loss in developing nestlings?).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Source/exposure level</td>
			<td>Intensity (lux)</td>
		</tr>
		<tr>
			<td>Full sunlight</td>
			<td>103000</td>
		</tr>
		<tr>
			<td>Full moonlight</td>
			<td>0.05&#8211;1</td>
		</tr>
		<tr>
			<td>Urban Sky glow</td>
			<td>0.2&#8211;0.5</td>
		</tr>
		<tr>
			<td>Exposure of free-living European blackbirds</td>
			<td>0.2 (0.07&#8211;2.2)</td>
		</tr>
		<tr>
			<td>Past experimental studies using the system</td>
			<td>1&#8211;3</td>
		</tr>
		<tr>
			<td>LED street lights</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>Low pressure sodium street lights</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>High pressure sodium</td>
			<td>~10</td>
		</tr>
		<tr>
			<td>Florescent lighting</td>
			<td>300</td>
		</tr>
		<tr>
			<td>Metal halide</td>
			<td>400&#8211;2000</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristic light intensities in the environment3,9, exposure levels of free-ranging birds41, and intensities used in past studies using this system (references in Table 2).
3. Implementing the exposure to ALAN
<ol>
	<li>Habituate the animals to the experimental setup.
	<ol>
		<li>If possible within the context of the experiment, habituate animals to the setup by placing dummy boxes on the top of the nest boxes at least 1 day prior to the experiment to minimize the effects of novelty aversion.</li>
	</ol>
	</li>
	<li>Survey the focal individuals.
	<ol>
		<li>Fit animals in the study population with&#160;passive integrative transponder (PIT) tags to allow for identification within nest boxes without disturbing the birds.</li>
		<li>In experiments involving the effect of ALAN on sleep behavior, visit the nest boxes on the night before the experiment and scan the boxes with a radio-frequency identification (RFID) reader to determine which birds are roosting inside.</li>
		<li>In experiments during the breeding season involving exposure of developing nestlings to ALAN, consistently monitor (e.g., every other day) nest boxes, and check for nest contents and adult identity. Carefully select nest boxes containing broods with certain characteristics (i.e., modal brood size, both parents present&#160;and feeding) for use in the experiment.</li>
	</ol>
	</li>
	<li>Select and implement the experiment.
	<ol>
		<li>For experiments involving sleep behavior, implement a repeated measures design by first recording individuals sleeping under conditions of darkness for at least one night to record undisturbed sleep in the absence of ALAN (control treatment) following steps 3.3.2-3.3.21.</li>
		<li>To this end, make sure to synchronize the time on the IR cameras with the local time prior to taking them into the field.</li>
		<li>Insert an SD card into the SD slot in&#160;the mini DVR recorder adjacent to the battery (Figure 2B; Supplementary Figure 2). Check to make sure that the SD card is empty, and if not, erase the data it contains.</li>
		<li>At least 2 h prior to the onset of darkness, remove the dummy box from on top of the nest box.</li>
		<li>Open the nest box lid.</li>
		<li>Place the plate containing the IR camera inside the nest box with the camera objective oriented downward.</li>
		<li>Extend the electronic connectors out of the grove in the nest box.</li>
		<li>Close the nest box lid.</li>
		<li>Place the enclosure containing the battery, recorder, and timer on top of the nest box.</li>
		<li>Connect the battery power connectors. Connect the red connector from the recorder to the white connector from the camera (audio), the yellow connector from the recorder to the yellow connector from the camera (video), and the black connector from the battery to the red connector from the camera (power) (Supplementary Figure 1 and Supplementary Figure 2).</li>
		<li>Push the record button to initiate the camera recording. 
		NOTE: The timer will not be set and/or the power will not be connected to the timer controlling the LEDs so that no ALAN will be produced on control nights.</li>
		<li>Check with a small tft screen to ensure the recording has started and that the image is correct. A port to connect the tft screen is located below the recorder (Supplementary Figure 2).</li>
		<li>Approximately 1 h after dark, return to the nest box and check the identity of the bird sleeping inside by moving a RFID transponder reader around the bottom and sides of the nest box and recording the unique identification number communicated from the PIT tag.</li>
		<li>On the morning following the control recording, at least 2 h after sunrise, return to the nest box and collect the battery system and IR camera.</li>
		<li>Again, place a dummy box on top of the nest box.</li>
		<li>In the laboratory or office, charge the battery and remove and download the SD card from the recorder to collect the behavioral data. 
		NOTE: Batteries have a life span of ~30 h in cold conditions to enable recording for the entire night, but need to be fully recharged between consecutive nights of recording.</li>
		<li>After successfully downloading the data, erase the data from the SD card and then reinsert it into the mini DVR recorder.</li>
		<li>On the subsequent night, implement the light exposure treatment (e.g., 1-3 lux, as used in past experiments using the system; Table 1 and Table 2).</li>
		<li>Set the timer system for the desired time period of light exposure.</li>
		<li>Follow the same steps (3.3.2-3.3.17) described above for the control recording, but also connect the timer to the power and the LEDs to the timer (Supplementary Figure 1 and Supplementary Figure 2).</li>
		<li>If desired, repeat the control recording (of sleep behavior under conditions of darkness, i.e., absence of ALAN) on night three.</li>
		<li>For experiments involving exposure of nestlings to ALAN, use control and experimental broods as described in steps 3.3.23-3.3.25.</li>
		<li>Place dummy boxes (lacking electronics) on top of the nest boxes of control broods and handle both control and experimental nestlings in equivalent ways.</li>
		<li>Implement the experimental ALAN exposure for experimental boxes. During the experimental period, mount the LED system and IR camera within the nest box, as described above, and set the timer to control the desired period of light exposure.</li>
		<li>Recharge the batteries. For experiments involving multiple nights of light exposure and video recording, collect the systems each morning to recharge the batteries during the day and then replace the system in the evening.</li>
	</ol>
	</li>
	<li>Collect data on the response variable(s) of interest.
	<ol>
		<li>If behavior within the nest box is the variable of interest, the IR camera will allow simultaneously documenting behavior (e.g., sleep behavior; Figure 3).</li>
		<li>Collect any other data of interest via additional monitoring methods, with sampling occurring at variable points in time (e.g., blood samples taken before and after light exposure15).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63381/63381fig03.jpg" /> 
Figure 3: Infrared image of a great tit inside a nest box exposed to ALAN. (A) Sleeping and (B) Alert great tit <a href="https://www.jove.com/files/ftp_upload/63381/63381fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The authors declare that they have no conflicts of interest.

This nest box-based system of LED lights and a paired IR camera has allowed researchers to assess a range of intriguing questions regarding the biological effects of ALAN. Moreover, there are many more research directions that can be pursued with the system. In addition, expanding the use of the system to other species could help foster an understanding of interspecific differences in sensitivity to ALAN. Below some non-exhaustive possibilities for future research are presented in the hope that this paper will help motiv...

Animals have evolved with the natural patterns of light and darkness that define day and night. Thus, circadian rhythms in hormonal systems orchestrate rest and activity patterns and allow animals to maximize fitness1,2,3. For instance, the circadian rhythm in glucocorticoid hormones, with a peak at the onset of daily activity, primes vertebrates to behave appropriately across the 24-h period via effects on glucose metabolism and responsiveness to environmental stressors4. Similarly, the pineal hormone melatonin, which is released in response to darkness, is integrally involved in governing patterns of circadian rhythmicity and also has antioxidant properties5,6. Entrainment of many aspects of circadian rhythmicity, such as melatonin release, is affected by the photoreception of levels of light in the environment. Thus, the introduction of artificial light into the environment to support human activity, recreation, and infrastructure has the potential to have wide-reaching effects on the behavior, physiology and fitness of free-ranging animals7,8. Indeed, diverse effects of exposure to artificial light at night (ALAN) have been documented9,10, and ALAN has been highlighted as a priority for global change research in the 21st century10.
Measuring the effects of ALAN on free-ranging animals poses non-trivial challenges for a number of reasons. First, mobile animals moving through the environment constantly experience different levels of light. Thus, how does one quantify the level of light that individual animals are exposed to? Even if levels of light on the territory of the animal can be quantified, the animal may employ avoidance strategies that affect patterns of exposure, thus demanding simultaneous tracking of animal location and light levels. Indeed, in most field studies, the mean and variation in light exposure levels are unknown11. Second, exposure to ALAN is often correlated with exposure to other anthropogenic disturbance factors, such as noise pollution, chemical exposure, and habitat degradation. For instance, animals occupying habitats along the margins of roadways will be exposed to light from street lamps, noise from vehicular traffic, and air pollution from vehicular emissions. How then does one effectively isolate the effects of ALAN from the effects of confounding variables? Rigorous field experiments that enable good measurements of both light exposure levels and response variables are essential to evaluating the severity of the biological effects of ALAN, and to developing effective mitigation strategies11.
This article describes an experimental approach that, although not without its limitations (see discussion section), helps assuage, if not eliminate the difficulties identified above. The approach entails experimentally manipulating ALAN levels inside the nest boxes of a free-living, diurnal bird species, the great tit (Parus major), using a system of light-emitting diode (LED) lights and an infrared (IR) camera installed within nest boxes. The setup enables simultaneous acquisition of video recordings, including audio, which allows researchers to assess effects on behaviors and vocalizations. Great tits utilize nest boxes for breeding, and sleep in the nest boxes between November and March. Females also sleep inside the nest boxes during the breeding season12. The system has also been used to a lesser extent to study effects of ALAN on blue tits (Cyanistes caeruleus). The first difficulty, involving knowing light levels encountered by the animal, is mitigated in that, given that an individual is willing to enter the nest box (or is already in the nest box in the case of immobile nestlings), light levels can be precisely determined by the researcher. The second difficulty, involving correlations to confounding variables, can be controlled by using nest boxes in similar environments, and/or measuring the levels of confounding variables near nest boxes. In addition, in cavity-nesting birds, adopting an experimental approach is powerful because nest boxes or natural cavities can shield nestlings and adults from ALAN13, which may explain why some correlative studies find little effect of ALAN (or anthropogenic noise)14, whereas experimental studies more often find clear effects (see below). Moreover, a repeated measures experimental design can be adopted in which individuals serve as their own control, which further increases statistical power, and the probability of detecting meaningful biological effects. The sections below: (1) explain the details of the design and implementation of the system, (2) summarize the important results that have been thus far derived using the system, and (3) propose future research directions that could be pursued, both in tits and other animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Broad spectrum; 15 mm x 5 mm; LED headlight</td>
			<td>RANEX; Gilze; Nederlands</td>
			<td>6000.217</td>
			<td>A similar model could also be used</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>BYD</td>
			<td>R1210A-C</td>
			<td>Fe-battery 12 V 120 Wh ( lithium iron phosphate battery)</td>
		</tr>
		<tr>
			<td>Dark green paint</td>
			<td></td>
			<td></td>
			<td>Optional. To color nest boxes/electronic enclosures</td>
		</tr>
		<tr>
			<td>Electrical tape</td>
			<td></td>
			<td></td>
			<td>For electronics</td>
		</tr>
		<tr>
			<td>Homemade timer system</td>
			<td>Amazon</td>
			<td>YP109A 12V</td>
			<td>A similar model could also be used</td>
		</tr>
		<tr>
			<td>Infrared camera</td>
			<td>Koberts-Goods, Melsungen, DE</td>
			<td>205-IR-L</td>
			<td>Mini camera; a similar model could also be used</td>
		</tr>
		<tr>
			<td>Light level meter</td>
			<td>ISO-Tech ILM; Corby; UK</td>
			<td>1335</td>
			<td>To calibrate light intensity</td>
		</tr>
		<tr>
			<td>Mini DVR video recorder</td>
			<td>Pakatak, Essex, UK</td>
			<td>MD-101</td>
			<td>Surveillance DVR Recorder Mini SD Car DVR with 32 GB</td>
		</tr>
		<tr>
			<td>Passive integrated transponder (PIT) tags</td>
			<td>Eccel Technology Ltd, Aylesbury, UK</td>
			<td>EM4102</td>
			<td>125 Kh; Provides unique electronic ID</td>
		</tr>
		<tr>
			<td>Radio frequency identification (RFID) Reader</td>
			<td>Trovan, Aalten, Netherlands</td>
			<td>GR-250</td>
			<td>To scan PIT tags and determine bird identity</td>
		</tr>
		<tr>
			<td>Resistor</td>
			<td>RS Components</td>
			<td></td>
			<td>Value depending on voltage battery and illumination</td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk</td>
			<td></td>
			<td>64 GB or larger</td>
		</tr>
		<tr>
			<td>SongMeter</td>
			<td>Wildlife Acoustics; Maynard, MA</td>
			<td></td>
			<td>Optional. Provides a means of monitoring vocalizations outside of nest boxes</td>
		</tr>
		<tr>
			<td>TFT Color LED Portable Test Monitor</td>
			<td>Walmart</td>
			<td></td>
			<td>Allows verification that the camera is on and recording the image correctly</td>
		</tr>
		<tr>
			<td>Wood</td>
			<td></td>
			<td></td>
			<td>To construct nest boxes/electronic encolsures</td>
		</tr>
	</tbody>
</table>

an experimental approach to investigating effects of artificial light at night on free-ranging animals: implementation, results, and directions for future research

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from human infrastructure and recreational activity. Artificial light at night (ALAN) has the potential to have widespread effects on animal behavior, physiology, and fitness, which can translate into broader-scale effects on populations and communities. Understanding the effects of ALAN on free-ranging animals is non-trivial due to challenges such as measuring levels of light encountered by mobile organisms and separating the effects of ALAN from those of other anthropogenic disturbance factors. Here we describe an approach that allows us to isolate the effects of artificial light exposure on individual animals by experimentally manipulating light levels inside nest boxes. To this end, a system can be used consisting of light-emitting diode (LED) light(s) adhered to a plate and connected to a battery and timer system. The setup allows exposure of individuals inside nest boxes to varying intensities and durations of ALAN while simultaneously obtaining video recordings, which also include audio. The system has been used in studies on free-ranging great tits (Parus major) and blue tits (Cyanistes caeruleus) to gain insight into how ALAN affects sleep and activity patterns in adults and physiology and telomere dynamics in developing nestlings. The system, or an adaptation thereof, could be used to answer many other intriguing research questions, such as how ALAN interacts with other disturbance factors and affects bioenergetic balance. Furthermore, similar systems could be installed in or near the nest boxes, nests or burrows of a variety of species to manipulate levels of ALAN, evaluate biological responses, and work towards building an interspecific perspective. Especially when combined with other advanced approaches for monitoring the behavior and movement of free-living animals, this approach promises to yield ongoing contributions to our understanding of the biological implications of ALAN.

The peer-reviewed research articles published using this system are summarized in Table 2. Several other manuscripts are in progress. These studies address three major suites of research questions. First, the system has been used to study the effects of light exposure on sleep behavior and activity levels in adults. To this end, a repeated measures experimental design was employed, in which the same individual was first recorded sleeping under natural conditions and subsequently recorded sleeping in a li...

Watch this Scientific Journal Video about An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Res

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from ...

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2.3K Views.  University of Antwerp. Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Video: An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Testing Nicotine Tolerance in Aphids Using an Artificial Diet Experiment

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well know to have insecticidal properties. One response of tobacco plants to herbivory, or being gnawed upon by insects, is to increase the production of this neurotoxic alkaloid. Here, we will demonstrate how to set up an experiment to address this question of whether a tobacco-adapted strain of the green peach aphid, Myzus persicae, can tolerate higher levels of nicotine than the a strain of this insect that does not infest tobacco in the field.

In this video and assay is demonstrated that tests the tolerance of nicotine to two types of aphids one that infests tobacco plants in the field and one that does not.

Plants may upregulate the production of many different seconday metabolites in response to insect feeding. One of these metabolites, nicotine, is well ...

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.

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.

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 ...

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 ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

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 ...

Experimental Approach to Examine Leptin Signaling in the Carotid Bodies and its Effects on Control of Breathing

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The carotid bodies (CB), a key organ of peripheral hypoxic sensitivity, express the long functional isoform of leptin receptor (LepRb) but the role of leptin signaling in control of breathing has not been fully elucidated. We examined the hypoxic ventilatory response (HVR) (1) in C57BL/6J mice before and after leptin infusion at baseline and after CB denervation; (2) in LepRb-deficient obese db/db mice at baseline and after LepRb overexpression in CBs. In C57BL/6J mice, leptin increased HVR and effects of leptin on HVR were abolished by CB denervation. In db/db mice, LepRb expression in CB augmented the HVR. Therefore, we conclude that leptin acts in CB to augment responses to hypoxia.

Our study focuses on the effects of leptin signaling in carotid body (CB) on the hypoxic ventilatory response (HVR). We performed &#39;loss of function&#39; experiments measuring the effect of leptin on HVR after CB denervation and &#39;gain of function&#39; experiments measuring HVR after overexpression of the leptin receptor in CB.

An adipocyte-produced hormone leptin is a potent respiratory stimulant, which may play an important role in defending respiratory function in obesity. The ...

Development of an Individual-Tree Basal Area Increment Model using a Linear Mixed-Effects Approach

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots located in Xinjiang Province, northwest China. To prevent high correlations among observations from the same sampling unit, we developed the model using a linear mixed-effects approach with random plot effect to account for stochastic variability. Various tree- and stand-level variables, such as indices for tree size, competition, and site condition, were included as fixed effects to explain the residual variability. In addition, heteroscedasticity and autocorrelation were described by introducing variance functions and autocorrelation structures. The optimal linear mixed-effects model was determined by several fit statistics: Akaike&#8217;s information criterion, Bayesian information criterion, logarithm likelihood, and a likelihood ratio test. The results indicated that significant variables of individual-tree basal area increment were the inverse transformation of diameter at breast height, the basal area of trees larger than the subject tree, the number of trees per hectare, and elevation. Furthermore, errors in variance structure were most successfully modeled by the exponential function, and the autocorrelation was significantly corrected by first-order autoregressive structure (AR(1)). The performance of the linear mixed-effects model was significantly improved relative to the model using ordinary least squares regression.

Mixed-effects models are flexible and useful tools for analyzing data with a hierarchical stochastic structure in forestry and could also be used to significantly improve the performance of forest growth models. Here, a protocol is presented that synthesizes information relating to linear mixed-effects models.

Here, we developed an individual-tree model of 5-year basal area increments based on a dataset including 21898 Picea asperata trees from 779 sample plots ...