This work was supported by a research grant from the German Federal Ministry of Education and Research (BMBF) (grant #13GW0043C) and and a European Office of Aerospace Research and Development (EOARD, grant # FA9550-15-1-0443)

1. RPE Cell Culture
<ol>
	<li>Isolation of RPE cells from porcine eyes
	<ol>
		<li>Obtain freshly enucleated porcine eyes from the local slaughterhouse. Keep them cool (4 &#176;C) and in a dark environment.</li>
		<li>Remove extracellular tissues with scissors and soak the eyes in an antiseptic solution for 5 min.</li>
		<li>Place the eyes in sterilized phosphate-buffered saline without calcium and magnesium (PBS (-)) until use.</li>
		<li>Using a scalpel, penetrate the sclera at about 5 mm posterior to the corneal limbus. Resect the whole anterior part of the eye by cutting with scissors all way through, parallel to the corneal limbus.</li>
		<li>Remove the anterior part of the eye (i.e., the cornea and lens) and the vitreous. Add 1 mL of PBS (-) and gently remove the neural retina. 
		NOTE: This &#34;eye cup,&#34; consisting of the sclera, choroid, and RPE, is now ready.</li>
		<li>Add pre-warmed (37 &#176;C) 0.25% trypsin in PBS (-) to the eye cup. Adjust the volume such that about 80% of the eye cup is filled with this trypsin solution.</li>
		<li>Incubate the eye cup with the trypsin solution in a 5% CO2 incubator at 37 &#176;C for 10 min.</li>
		<li>Remove the eye cup from the incubator and replace the 0.25% trypsin solution with a PBS (-) solution with 0.05% trypsin + 0.2% ethylenediaminetetraacetic acid tetrasodium salt (EDTA&#183;4N). Incubate the eye cup in the incubator for 45 min. 
		NOTE: After 45 min, the RPE cells will be either just loosely attached to the Bruch&#39;s membrane or already detached and floating in the trypsin-EDTA solution.</li>
		<li>Collect the RPE cells by gentle pipetting. Collect the cells and solution in a conical tube filled with 10 mL of culture medium (DMEM high glucose with L-glutamine), including 10% porcine serum, antibiotic/antimycotic, and sodium pyruvate (1 mM). 
		NOTE: The serum may neutralize the effect of trypsin.</li>
		<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and add 10 mL of fresh medium. Centrifuge again under the same conditions for 5 min.</li>
		<li>Remove the supernatant and add new medium, such that the cell concentration results in 5 x 105cells/mL (determined by counting the cells using a hemocytometer). Mix well by gentle pipetting.</li>
		<li>Distribute the cell suspension in cell culture dishes. Use 3 mL per 60 mm-diameter culture dish. 
		NOTE: This culture is called passage zero (P0).</li>
		<li>Maintain the cells in a 5% CO2 incubator at 37 &#176;C. Change half of the conditioned medium to fresh medium every second day.</li>
		<li>Subculture (step 1.2) if it becomes confluent.</li>
	</ol>
	</li>
	<li>Subculture of the RPE cell culture
	<ol>
		<li>Remove the culture medium and rinse the cells twice with PBS (-).</li>
		<li>Incubate the cells with PBS (-) solution with 0.05% trypsin + 0.2% EDTA in a 5% CO2 incubator at 37 &#176;C for 5 min.</li>
		<li>Detach the RPE cells by gentle pipetting and collect the cell suspension in a conical tube filled with 10 mL of culture medium, including 10% porcine serum.</li>
		<li>Centrifuge the cell suspension at 400 x g for 5 min at room temperature.</li>
		<li>Remove the supernatant and add new culture medium, making the cell concentration 5 x 105 cells/mL (determined by counting the cell number with a hemocytometer). Distribute the cells in new 60 mm-diameter culture dishes, as described in step 1.1.13. 
		NOTE: The cell culture is now passage 1 (P1).</li>
		<li>After confluency is reached, subculture the P1 culture to P2, using the same procedure described in steps 1.2.1-1.2.5. From the P2 culture, seed the cells on smaller culture dishes (30-mm inner diameter) instead of 60 mm-diameter culture dishes.</li>
		<li>For the experiments, use P2 or P3 cultures.</li>
	</ol>
	</li>
</ol>
2. Thulium Laser Irradiation
<ol>
	<li>Construction of the irradiation station
	<ol>
		<li>Connect a thulium laser device (1.94 &#181;m, power range: 0-20 W) to a 0.22-NA, 365-&#181;m core diameter fiber.</li>
		<li>Mechanically fix the fiber tip to the metal arm that is horizontally fixed to the vertical metal post of the irradiation station. Place the vertical post such that the tip of the laser fiber is located above the hot plate on which the cell culture dish is to be placed during irradiation.</li>
		<li>Lay a white paper on the hot plate and turn the aiming beam on (&#955; = 635 nm, max = 1 mW, diameter at paper level &#8776; 30 mm). Mark the circumference of the aiming beam on the white paper so that the position where the culture dish is to be placed during irradiation is known. 
		NOTE: The z-plane of the fiber tip may be changeable. Without any additional imaging optics, the laser spot diameter on the cell culture plane, placed 12 cm below the fiber tip, is about 30 mm, which is almost equivalent to the inner diameter of the cell culture dish. A schematic drawing of the setup is shown in Figure 1.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54326/54326fig1.jpg" /> 
Figure 1: Schematic Image of the Thulium Laser Irradiation Station. A culture dish is placed on the heating plate. The cells are placed 12 cm below the thulium laser fiber tip so that the beam size is almost identical to the inner diameter of the culture dish (about 30 mm). The laser irradiation procedure is controlled by a time-controlled routine of the custom-made system design platform. The power setting must be determined before the irradiation program is started. <a href="http://cloudfront.jove.com/files/ftp_upload/54326/54326fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Laser irradiation of the cell culture
	<ol>
		<li>1 h before irradiation, replace the culture medium completely with 1.2 mL of fresh medium. 
		NOTE: This is a CRITICAL STEP and must be strictly followed.</li>
		<li>Place the irradiation station (i.e., the hot plate and the post with which to fix the laser fiber) on a clean bench.</li>
		<li>Remove the cell culture dish from the incubator and place it on the marked position on the hot plate (step 2.1.3).</li>
		<li>Wear protective glasses. Turn on the thulium laser. Set the power as desired on the laser device (tunable from 0 - 20 W). Turn the emission on.</li>
		<li>Start a system design platform that controls the laser irradiation and timing protocol (supplemental file).</li>
		<li>Immediately after placing the culture dish on the hot plate, click the &#34;pre-heating time&#34; to start the timer for 140 s (&#34;pre-heating time 1&#34;); this will keep the culture medium temperature at 37 &#176;C before irradiation. 
		NOTE: After 140 s, a beep sound will turn on, and the next timer (&#34;pre-heating time 2&#34;) will start automatically counting 8 s. During this 8 s, the examiner may open the culture dish. After 148 s of pre-heating, a 10 s-long laser irradiation on the cell culture will be conducted automatically. In case of emergency, equip the laser device with a force-quit button to stop the laser immediately. This is a CRITICAL STEP and must be strictly followed. A special point of caution relates to opening the cover of the dish just before irradiation, at the beginning of the 8 s pre-heating time. Opening the cover may cool down the medium surface very quickly.</li>
		<li>After irradiation, immediately place the cover back onto the culture dish, leave the culture dish on the hot plate for an additional 7 s, and place it back in the 5% CO2 incubator at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Measurement of the temperature distribution at the cellular level (temperature calibration)
	<ol>
		<li>Make small holes (about 300 &#181;m in diameter) close to the bottom on four sides (every 90&#176;) of a 30 mm diameter culture dish (without cells); use the tip of a needle (20G) heated with a Bunsen burner. Seal the holes with electrical isolation tape from outside and make a small hole with a fine needle so that only a fine thermocouple (200 &#181;m in diameter) may be inserted through this hole under watertight conditions.</li>
		<li>At the outside of the culture dish bottom, draw 2 perpendicular diameters and set the crossing point (i.e., the center of the bottom side) as the coordinate zero (0). Mark every 3 mm radially to the outside out the dish (i.e., 0, 3, 6, 9, 12, and 15 mm) in each direction along the lines (Figure 2, blue dots); the number of points should be 21 in total.</li>
		<li>Fill the cell culture dish with 1.2 mL of new culture medium. Place the culture dish onto a hot plate at 37 &#176;C, insert a fine thermocouple (200 &#181;m in diameter) into the side hole, and place its sensitive tip onto a marked position to be measured.</li>
		<li>Wear protective glasses. Turn on the thulium laser and manually set the power (between 0 and 20 W, in 0.1-W increments) of the laser devise. 
		NOTE: For temperature calibration, measurements with the power in increments of 3 W should be sufficient.</li>
		<li>Turn on the system design platform and click the &#34;Start Temp. Acquisition&#34; button (supplemental file) to start the temperature measurement.</li>
		<li>Conduct same procedure as in step 2.2.6. 
		NOTE: The control program measures the temperature of the inserted thermocouple every 100 ms and shows the temperature progression during irradiation in the GUI.</li>
		<li>Conduct these procedures for all 21 measurement points and at different power settings. Repeat the whole procedure three times for all points and for all power settings to achieve reliable data.</li>
		<li>Export temperature data as csv data, which can eventually be converted to a spreadsheet. Average the maximal temperature at the end of irradiation for the triplicate measurements at every point. Average the values from the points on the same circle (4 points in total, except the central point).</li>
		<li>Plot the obtained averaged maximal temperature on a graph, making the distance from the center of the dish (mm) as the x-axis and the temperature increase (&#916;T, &#176;C) as the y-axis. Use the fit function of a mathematical software program to fit a Gaussian model to the raw data. Create a Gaussian fit temperature distribution.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54326/54326fig2.jpg" /> 
Figure 2: The Points for Temperature Calibration in One Cell Culture Dish. The temperature data was measured in the center and at 5 radial points over 4 different angles (blue dots). <a href="http://cloudfront.jove.com/files/ftp_upload/54326/54326fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Biological Assessments for Cell Responses after Different Thermal Irradiations
<ol>
	<li>Assessment of cell viability ( i.e., living, apoptotic, and dead) following different power settings and the determination of the cell death threshold
	<ol>
		<li>At the indicated time points (i.e., 3, 24, and 48 h after irradiation), wash the cells with PBS (-) and use a commercially available kit to assess cell viability (i.e., vital, apoptotic, dead) according to the manufacturer&#39;s protocol.</li>
		<li>Prepare a staining solution by adding 5 &#181;L of fluorescein isothiocyanate (FITC)-annexin V, 5 &#181;L of ethidium homodimer III, and 5 &#181;L of Hoechst 33342 to 100 &#181;L of 1x binding buffer (all are kit components). Prepare enough staining solution to cover the cells. Incubate the cells for 15 min.</li>
		<li>Wash the cell culture with the binding buffer twice, replace the binding buffer with PBS (-), and set the culture on the stage of a fluorescence microscope.</li>
		<li>Switch the light path to the ocular lens, select the 4&#39;,6-Diamidin-2-phenylindol (DAPI) filter, turn on the illumination light, and find the focused plane with the 4x objective.</li>
		<li>Change the light path to the camera, find the image on the computer screen in the microscope imaging software, and adjust the focus.</li>
		<li>Use the stitch function (i.e., the function to record multiple images across the dish and then create a single, large image) of the microscope-specific software to obtain the fluorescence image of the whole cell culture dish. Use 3 different filter sets-DAPI, FITC, and tetramethylrhodamine (TRITC)-to image Hoechst 33342-positive cells (all cell nuclei), FITC-annexin V-positive cells (apoptotic), and ethidium homodimer III-positive cells (dead), respectively.</li>
		<li>Measure the radius (mm) of the dead (ethidium homodimer III-positive) region and the outer/inner radius of the apoptotic (annexin V-positive) band-form region in the stained cell cultures. Apply these radii to the fitted Gaussian function of the temperature distribution for the corresponding power setting. Calculate the exact temperature at the rim of the dead or apoptotic region to clarify the threshold temperatures for cell death and apoptotic change.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Inagaki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+efficacy+of+pure+yellow+(577-nm)+and+810-nm+subthreshold+micropulse+laser+photocoagulation+combined+with+yellow+(561-577-nm)+direct+photocoagulation+for+diabetic+macular+edema.">Comparative efficacy of pure yellow (577-nm) and 810-nm subthreshold micropulse laser photocoagulation combined with yellow (561-577-nm) direct photocoagulation for diabetic macular edema.</a> Jpn J Ophthalmol. 59 (1), 21-28 (2015).</li><li>Roider, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+retina+therapy+(SRT)+for+clinically+significant+diabetic+macular+edema.">Selective retina therapy (SRT) for clinically significant diabetic macular edema.</a> Graefes Arch Clin Exp Ophthalmol. 248 (9), 1263-1272 (2010).</li><li>Brinkmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+temperature+determination+during+retinal+photocoagulation+on+patients.">Real-time temperature determination during retinal photocoagulation on patients.</a> J Biomed Opt. 17 (6), 061219 (2012).</li><li>Yoshimura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocoagulated+human+retinal+pigment+epithelial+cells+produce+an+inhibitor+of+vascular+endothelial+cell+proliferation.">Photocoagulated human retinal pigment epithelial cells produce an inhibitor of vascular endothelial cell proliferation.</a> Invest Ophthalmol Vis Sci. 36 (8), 1686-1691 (1995).</li><li>Denton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Damage+Thresholds+for+Exposure+to+NIR+and+Blue+Lasers+in+an+In+Vitro+RPE+Cell+System.">Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System.</a> Invest Ophthalmol Vis Sci. 47 (7), 3065-3073 (2006).</li><li>Shrestha, R., Choi, T. Y., Chang, W., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-precision+micropipette+sensor+for+cellular-level+real-time+thermal+characterization.">A high-precision micropipette sensor for cellular-level real-time thermal characterization.</a> Sensors (Basel). 11 (9), 8826-8835 (2011).</li><li>Gao, F., Ye, Y., Zhang, Y., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+bath+hyperthermia+reduces+stemness+of+colon+cancer+cells.">Water bath hyperthermia reduces stemness of colon cancer cells.</a> Clin Biochem. 46 (16-17), 1747-1750 (2013).</li><li>Jansen, E. D., van Leeuwen, T. G., Motamedi, M., Borst, C., Welch, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+dependence+of+the+absorption+coefficient+of+water+for+midinfrared+laser+radiation.">Temperature dependence of the absorption coefficient of water for midinfrared laser radiation.</a> Lasers Surg Med. 14 (3), 258-268 (1994).</li><li>Iwami, H., Pruessner, J., Shiraki, K., Brinkmann, R., Miura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+a+laser-induced+sub-lethal+temperature+rise+on+RPE+cells+from+oxidative+stress.">Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress.</a> Exp Eye Res. 124, 37-47 (2014).</li><li>Denton, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+correlated+microthermography+maps+threshold+temperature+in+laser-induced+damage.">Spatially correlated microthermography maps threshold temperature in laser-induced damage.</a> J Biomed Optics. 16 (3), (2011).</li><li>Morgan, C. M., Schatz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrophic+creep+of+the+retinal+pigment+epithelium+after+focal+macular+photocoagulation.">Atrophic creep of the retinal pigment epithelium after focal macular photocoagulation.</a> Ophthalmology. 96 (1), 96-103 (1989).</li></ol>

The authors have nothing to disclose.

In discussing temperature-related biological cellular responses, not only the temperature, but also the time duration of the increased temperature, is of importance, since most biochemical processes are time-dependent. Particularly in the field of laser-induced hyperthermia in ophthalmology, due to the short time range-from milliseconds to seconds-it is difficult to investigate cellular thermal effects with precise temperature control. Therefore, a laser irradiation setup suitable for the cell culture model and with an o...

Understanding temperature-dependent cell biological responses is of great importance to successful hyperthermia treatments. Retinal laser photocoagulation with a thermal laser, used in ophthalmology, is one of the most established laser treatments in medicine. Visible light, mostly from green to yellow wavelengths, is used in retinal laser treatment. The light is highly absorbed by the melanin in retinal pigment epithelial (RPE) cells, which form the outermost cell monolayer of the retina. There has been recent interest among physicians and researchers in very mild thermal irradiation (sub-visible photocoagulation) as a new therapeutic strategy for different kinds of retinal disorders1,2. Following this trend, our interest is in sub-lethally heating RPE cells under precise temperature control, a technique called temperature-controlled photothermal therapy (TC-PTT).
Recent optoacoustic technology from our institute has allowed for the real-time measurement of temperature increases at irradiated sites in the retina. This enables control over the temperature increase during irradiation3. However, since sub-lethal hyperthermia on the retina, caused by heating RPE cells sub-lethally, has not been previously considered due to the impossibility of measuring and controlling the temperature, the temperature-dependent cell responses of RPE cells following thermal laser irradiation has been studied very little to date. Moreover, not only has the temperature difference not been discussed in detail, but also the difference in the cell behavior of the surviving cells after sub-lethal and lethal irradiation. Therefore, to gather scientific evidence on TC-PTT-based treatments, we aim to elucidate the temperature-dependent RPE cell biological responses and their mechanisms using in vitro experimental setups.
For this purpose, it is necessary to establish a cell-heating setup that meets the following conditions: 1) a possibility for fast temperature increases, 2) a precisely controlled time and temperature, and 3) a relatively high number of examined cells for biological experiments. Regarding the heating method, a clinical laser, such as a frequency-doubled Nd.YAG laser (532 nm), is unfortunately unsuitable for cell culture heating. This is because of the strongly reduced number of melanosomes in cultured RPE cells. The laser light absorption might be inhomogeneous, and the temperature increase at the cellular level is variable between experiments, even when irradiated with same radiation power. Several previous studies have reported the use of black paper beneath the dish bottom during irradiation4 or the use of additional melanosomes that are phagocytized by the culture cells before the experiments5,6. Many of the in vitro biological studies to assess hyperthermia-induced cell responses have been performed using a hot plate, a water bath, or a CO2 incubator with a temperature setting7. These methods require a long heating period because it takes some time (i.e., several minutes) to reach the desired temperature. Furthermore, using these methods, it is difficult to obtain a detailed thermal history (i.e., temperature multiplied by time) at the cellular level. Moreover, the temperature among the cells at different positions in one culture dish may differ due to variable temperature diffusion. In most cases, this temporal and spatial temperature information during hyperthermia has not been taken into consideration for biological analyses, even though biological cell response may be critically affected by the temperature and the time duration of the increased temperature.
To overcome these problems, a continuous-wave thulium laser was used here to heat the cells. Thulium laser radiation (&#955; = 1.94 &#181;m) is strongly absorbed by water8, and the cells at the bottom of the culture dish are thermally stimulated solely through thermal diffusion. The laser fiber with a 365-&#181;m diameter is set about 12 cm above the culture dish, without any optics in between. The laser beam diameter diverges such that it is almost equivalent to the inner diameter of the culture dish (30 mm) at the surface of the culture medium.With a consistent amount of culture medium, it is possible to irradiate the cells with the temperature increase of high repeatability. Variable power settings enable irradiation with up to 20 W, and the medium temperature at the cellular level may be increased up to &#916;T &#8776; 26 &#176;C in 10 s.
By modifying the irradiation conditions, it is also possible to change the laser beam profile to vary the temperature distribution in a culture dish. For example, it is possible to investigate with a Gaussian-like temperature distribution, as in the current study, or with a homogeneous temperature distribution. The latter may be advantageous for investigating the effects of temperature-dependent cell responses more specifically for sub-lethal temperature increases, but not for cell death stress or wound healing responses.
Altogether, thulium laser irradiation may enable the investigation of different kinds of biological factors, such as gene/protein expression, cell death kinetics, cell proliferation, and cell functionality development, after different thermal exposures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle's Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5796-500ML</td>
			<td>Add (2)-(4) before use. Warm in 37&#176;C water bath before use.</td>
		</tr>
		<tr>
			<td>Antibiotic Antimycotic Solution (100&#215;)</td>
			<td>Sigma-Aldrich</td>
			<td>A5955-100ML</td>
			<td>Containing 10000 units penicillin, 10 mg streptomycin and 25 &#956;g Amphotericin B in 1ml. Add 5.5 ml in 500 ml medium bottle (1) before use.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100 mM)</td>
			<td>Sigma-Aldrich</td>
			<td>S8636-100ML</td>
			<td>Add 5.5 ml in 500 ml medium bottle (1) before use (final concentration: 1 mM)</td>
		</tr>
		<tr>
			<td>Porcine serum</td>
			<td>Sigma-Aldrich</td>
			<td>12736C-500ML</td>
			<td>Add 50 ml in 500 ml medium bottole (1) before use (final: 10%)</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>T4799-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>ED-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF Quantikine ELISA Kit</td>
			<td>R&#38;D System</td>
			<td>DVE00</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxiselect Total Glutathione Assay Kit</td>
			<td>Cell Biolabs, Inc</td>
			<td>STA-312</td>
			<td></td>
		</tr>
		<tr>
			<td>Apoptotic/Necrotic/Healthy Cells Detection Kit</td>
			<td>PromoKine</td>
			<td>PK-CA707-30018</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thulium laser</td>
			<td>Starmedtec GmbH</td>
			<td>Prototype</td>
			<td>0-20 W</td>
		</tr>
		<tr>
			<td>365 mm core diameter fiber</td>
			<td>LASER COMPONENTS Germany</td>
			<td>CF01493-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocouple</td>
			<td>Omega Engineering Inc</td>
			<td>HYP-0- 33-1-T-G-60-SMPW-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>MEDAX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader (spectrofluorometer)</td>
			<td>Molecular Device</td>
			<td>Spectramax M4</td>
			<td></td>
		</tr>
		<tr>
			<td>cell homogenizer</td>
			<td>QIAGEN</td>
			<td>TissueLyser LT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ti</td>
			<td></td>
		</tr>
		<tr>
			<td>mathematical software program</td>
			<td>The Mathworks. Inc</td>
			<td>MATLAB Release 2015b</td>
			<td></td>
		</tr>
		<tr>
			<td>system-design platform</td>
			<td>National Instrument</td>
			<td>Labview</td>
			<td>Laboratory Virtual Instrument Engineering Workbench</td>
		</tr>
	</tbody>
</table>

continuous-wave thulium laser for heating cultured cells to investigate cellular thermal effects

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 &#181;m is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase.
To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time.
The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.

Temperature distribution after different power settings
All temperature developments for each single irradiation were monitored in the temperature calibration. From this data, the maximal temperature at the measured point was obtained and defined as Tmax (&#176;C). As shown in Figure 3A, the program was executed at the time point when the culture dish was placed on the heating plate. After t...

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Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

An original experimental setup for heating cells in a culture dish using 1.94 &#181;m continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

An original method to heat cultured cells using a 1.94 &#181;m continuous-wave thulium laser for biological assessment is introduced here. Thulium laser ...

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7.7K Views.  University of Luebeck. An original experimental setup for heating cells in a culture dish using 1.94 µm continuous-wave laser radiation is introduced here. Using this method, the biological responses of retinal pigment epithelial (RPE) cells after different thermal exposures can be investigated.

Video: Continuous-wave Thulium Laser for Heating Cultured Cells to Investigate Cellular Thermal Effects

Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and cardiovascular disease1-11. Traditional cell biological approaches to studying cellular function involve culturing cells on a rigid substratum (plastic dishes or glass coverslips) which cannot account for the effect of an elastic ECM or the variations in ECM stiffness between tissues. To model in vivo tissue compliance conditions in vitro, we and others use ECM-coated hydrogels. In our laboratory, the hydrogels are based on polyacrylamide which can mimic the range of tissue compliances seen biologically12. "Reactive" cover slips are generated by incubation with NaOH followed by addition of 3-APTMS. Glutaraldehyde is used to cross-link the 3-APTMS and the polyacrylamide gel. A solution of acrylamide (AC), bis-acrylamide (Bis-AC) and ammonium persulfate is used for the polymerization of the hydrogel. N-hydroxysuccinimide (NHS) is incorporated into the AC solution to crosslink ECM protein to the hydrogel. Following polymerization of the hydrogel, the gel surface is coated with an ECM protein of choice such as fibronectin, vitronectin, collagen, etc.
The stiffness of a hydrogel can be determined by rheology or atomic force microscopy (AFM) and adjusted by varying the percentage of AC and/or bis-AC in the solution12. In this manner, substratum stiffness can be matched to the stiffness of biological tissues which can also be quantified using rheology or AFM. Cells can then be seeded on these hydrogels and cultured based upon the experimental conditions required. Imaging of the cells and their recovery for molecular analysis is straightforward. For this article, we define soft substrata as those having elastic moduli (E) &lt;3000 Pascal and stiff substrata/tissues as those with E &gt;20,000 Pascal.

The effect of substrata stiffness on cellular function can be modeled in vitro using polyacrylamide hydrogels of varying compliances.

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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 ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

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 ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...