The authors have no acknowledgements.

1. Preparation of Cells
<ol>
	<li>Plate 20,000 cells in 2 mL of culture medium into each well of a 6-well plate using sterile technique in a biosafety cabinet.</li>
	<li>Place 6-well plates in a humidified incubator (37 &#176;C, 5% CO2) for 24 h to allow the cells to adhere to plates.</li>
</ol>
2. Preparation of Photosensitizer for Treatment of Cells
<ol>
	<li>Prepare 0.5, 1, and 2 mM 5-ALA solutions in culture medium. If fetal bovine serum (FBS) is an ingredient of culture medium, prepare and treat cells with 5-ALA in culture medium solution with 0.1% FBS for incubations.11,12 In this case, SCC-13 cells do not require FBS, so 5-ALA was added directly to keratinocyte serum-free medium supplemented with bovine pituitary extract and epidermal growth factor.</li>
	<li>Aspirate the culture medium and wash the cells with at least 2 mL of phosphate buffered saline (PBS).</li>
	<li>Aspirate PBS and add 2 mL of 0, 0.5, 1, or 2 mM 5-ALA solutions into separate plates or wells.</li>
	<li>Incubate the cells with 0, 0.5, 1, or 2 mM 5-ALA on a 36 to 37 &#176;C heating block for 30 min. Protect the cells from light exposure during incubation using aluminum foil.</li>
	<li>Aspirate the 0, 0.5, 1, and 2 mM 5-ALA solutions and wash the cells with 2 mL of PBS.</li>
	<li>Aspirate PBS and add 2 mL of culture medium for blue light irradiation.</li>
</ol>
3. Blue Light Phototherapy
<ol>
	<li>Before irradiating the cells, turn on the blue light device (e.g., light-emitting diode, fluorescent, or halogen light) and run for one cycle (1,000 s) to warm up the device. The blue light device should have an output wavelength of 417 +/- 5 nm. Allowing the device to run for one cycle before treatments ensures that the blue light wavelength and irradiance are consistent throughout the photoactivation phase.</li>
	<li>Use a photometer to measure irradiance at the cell surface. The blue light irradiance at the cell surface should be 10 mW/cm2. The distance between the light and the cells may need to be adjusted to achieve an irradiance of 10 mW/cm2 depending on the intensity of the light source.</li>
	<li>Place 0, 0.5, 1, and 2 mM ALA treatment groups on a black surface under the blue light source. Irradiate the cells with blue light for 1,000 s (16 min and 40 s) for a total fluence of 10 J/cm2. Following blue light photoactivation, the cells are ready for analysis.</li>
</ol>
4. Collection and Staining
<ol>
	<li>Prepare flow buffer according to manufacturer guidelines (see Table of Materials).</li>
	<li>Aspirate culture medium and wash cells with 2 mL of PBS.</li>
	<li>Add 1 mL of 0.25% trypsin-EDTA and allow cells to detach for approximately 3 to 5 min. The cells may be examined under the microscope to confirm cell detachment.</li>
	<li>Add 1 mL of culture medium (or PBS) with 10% fetal bovine serum to deactivate the trypsin.</li>
	<li>Collect cell suspension into labeled 5 mL flow tubes.</li>
	<li>Spin 5 mL flow tube in a centrifuge at 201 x g for 5 min. Aspirate solution without removing cell pellet.</li>
	<li>Add 200 &#181;L of flow buffer with conjugated annexin-V antibody (1 &#181;L of annexin-v per 39 &#181;L of flow buffer) to each sample.</li>
	<li>Resuspend the cells and place in the incubator (37 &#176;C, 5% CO2) for 20 min to allow annexin-V binding.</li>
	<li>Add 3 &#181;L of 7-AAD to each sample. Incubate for 5 min at room temperature.</li>
</ol>
5. Flow Cytometric Analysis
<ol>
	<li>Follow flow cytometer manufacturer guidelines for set-up (see Table of Materials).</li>
	<li>Collect and analyze samples with flow cytometry.13</li>
	<li>Perform statistical comparison of treatment and control groups using analysis of variance (ANOVA)14. Compare the means of the treatment groups to the mean of the control with a Dunnett&#39;s test for post hoc analysis.</li>
</ol>

<ol>
	<li>Morton, C., 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=European+Dermatology+Forum+guidelines+on+topical+photodynamic+therapy.">European Dermatology Forum guidelines on topical photodynamic therapy.</a> European Journal of Dermatology. 25 (4), 296-311 (2015).</li><li>Ozog, D. M., 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=Photodynamic+therapy:+A+clinical+consensus+guide.">Photodynamic therapy: A clinical consensus guide.</a> Dermatologic Surgery: Official Publication for American Society for Dermatologic Surgery. 42 (7), 804-827 (2016).</li><li>Abrahamse, H., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+photosensitizers+for+photodynamic+therapy.">New photosensitizers for photodynamic therapy.</a> Biochemical Journal. 473 (4), 347-364 (2016).</li><li>Josefsen, L. B., Boyle, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+and+the+development+of+metal-based+photosensitisers.">Photodynamic therapy and the development of metal-based photosensitisers.</a> Metal-Based Drugs. 2008, 276109 (2008).</li><li>Ohgari, Y., 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=Mechanisms+involved+in+&#948;-aminolevulinic+acid+(ALA)-induced+photosensitivity+of+tumor+cells:+Relation+of+ferrochelatase+and+uptake+of+ALA+to+the+accumulation+of+protoporphyrin.">Mechanisms involved in &#948;-aminolevulinic acid (ALA)-induced photosensitivity of tumor cells: Relation of ferrochelatase and uptake of ALA to the accumulation of protoporphyrin.</a> Biochemical pharmacology. 71 (1-2), 42-49 (2005).</li><li>Kessel, D., Luo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+photodamage+and+PDT-induced+apoptosis.">Mitochondrial photodamage and PDT-induced apoptosis.</a> Journal of Photochemistry and Photobiology B: Biology. 42 (2), 89-95 (1998).</li><li>Goldberg, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+in+skin+rejuvenation.">Photodynamic therapy in skin rejuvenation.</a> Clinics in Dermatology. 26 (6), 608-613 (2008).</li><li>Sakamoto, F. H., Lopes, J. D., Anderson, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+acne+vulgaris:+A+critical+review+from+basics+to+clinical+practice:+Part+I.+Acne+vulgaris:+When+and+why+consider+photodynamic+therapy.">Photodynamic therapy for acne vulgaris: A critical review from basics to clinical practice: Part I. Acne vulgaris: When and why consider photodynamic therapy.</a> Journal of the American Academy of Dermatology. 63 (2), 183-193 (2010).</li><li>Boen, M., Brownell, J., Patel, P., Tsoukas, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+photodynamic+therapy+in+acne:+An+evidence-based+review.">The role of photodynamic therapy in acne: An evidence-based review.</a> American Journal of Clinical Dermatology. 18 (3), 311-321 (2017).</li><li>Allison, R. R., Moghissi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncologic+photodynamic+therapy:+Clinical+strategies+that+modulate+mechanisms+of+action.">Oncologic photodynamic therapy: Clinical strategies that modulate mechanisms of action.</a> Photodiagnosis and Photodynamic Therapy. 10 (4), 331-341 (2013).</li><li>Koo, E., Austin, E., Mamalis, A., Jagdeo, 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=Thermal+ultra+short+photodynamic+therapy:+heating+fibroblasts+during+sub-30-minute+incubation+of+5-aminolevulinic+acid+increases+photodynamic+therapy-induced+cell+death.">Thermal ultra short photodynamic therapy: heating fibroblasts during sub-30-minute incubation of 5-aminolevulinic acid increases photodynamic therapy-induced cell death.</a> Dermatologic Surgery: Official Publication for American Society for Dermatologic Surgery. , (2017).</li><li>Koo, E., Austin, E., Mamalis, A., Jagdeo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+ultra+short+sub-30+minute+incubation+of+5-aminolevulinic+acid+photodynamic+therapy+in+vitro.">Efficacy of ultra short sub-30 minute incubation of 5-aminolevulinic acid photodynamic therapy in vitro.</a> Lasers in Surgery and Medicine. 49 (6), 592-598 (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Biology.+Annexin+V+and+Propidium+Iodide+Labeling.">Cell Biology. Annexin V and Propidium Iodide Labeling.</a> JoVE. , (2018).</li><li>Mamalis, A., Koo, E., Sckisel, G., Siegel, D., Jagdeo, 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-dependent+impact+of+thermal+aminolaevulinic+acid+photodynamic+therapy+on+apoptosis+and+reactive+oxygen+species+generation+in+human+dermal+fibroblasts.">Temperature-dependent impact of thermal aminolaevulinic acid photodynamic therapy on apoptosis and reactive oxygen species generation in human dermal fibroblasts.</a> British Journal of Dermatology. 175 (3), 512-519 (2016).</li><li>Gilaberte, Y., 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=Cellular+intrinsic+factors+involved+in+the+resistance+of+squamous+cell+carcinoma+to+photodynamic+therapy.">Cellular intrinsic factors involved in the resistance of squamous cell carcinoma to photodynamic therapy.</a> Journal of Investigative Dermatology. 134 (9), 2428-2437 (2014).</li><li>Yoon, H. 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=Nanophotosensitizers+engineered+to+generate+a+tunable+mix+of+reactive+oxygen+species,+for+optimizing+photodynamic+therapy,+using+a+microfluidic+device.">Nanophotosensitizers engineered to generate a tunable mix of reactive oxygen species, for optimizing photodynamic therapy, using a microfluidic device.</a> Chemistry of Materials. 26 (4), 1592-1600 (2014).</li><li>Chen, Y. -. C., Lou, X., Zhang, Z., Ingram, P., Yoon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cancer+cell+sphere+formation+for+characterizing+the+efficacy+of+photo+dynamic+therapy+in+3D+cell+cultures.">High-throughput cancer cell sphere formation for characterizing the efficacy of photo dynamic therapy in 3D cell cultures.</a> Scientific Reports. 5, 12175 (2015).</li><li>Campos, C., Inada, N., Kurachi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+PDT+on+breast+cancer+spheroids.">Low-dose PDT on breast cancer spheroids.</a> Optical Methods for Tumor Treatment and Detection: Mechanisms and Techniques in Photodynamic Therapy XXVII. , (2018).</li><li>Yang, 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=The+influence+of+temperature+on+5-aminolevulinic+acid-based+photodynamic+reaction+in+keratinocytes+in+vitro.">The influence of temperature on 5-aminolevulinic acid-based photodynamic reaction in keratinocytes in vitro.</a> Photodermatology, Photoimmunology &amp; Photomedicine. 26 (2), 83-88 (2010).</li><li>Juzeniene, A., Juzenas, P., Kaalhus, O., Iani, V., Moan, 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+effect+on+Accumulation+of+protoporphyrin+IX+after+topical+application+of+5-aminolevulinic+acid+and+its+methylester+and+hexylester+derivatives+in+normal+mouse+skin.">Temperature effect on Accumulation of protoporphyrin IX after topical application of 5-aminolevulinic acid and its methylester and hexylester derivatives in normal mouse skin.</a> Photochemistry and Photobiology. 76 (4), 452-456 (2002).</li><li>Novak, B., Heesen, L., Schary, N., Lubbert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+different+illumination+parameters+on+protoporphyrin+IX+induced+cell+death+in+squamous+cell+carcinoma+cells.">The influence of different illumination parameters on protoporphyrin IX induced cell death in squamous cell carcinoma cells.</a> Photodiagnosis and Photodynamic Therapy. 21, 385-392 (2018).</li></ol>

DUSA/Sun Pharmaceutical provided 5-ALA and the BLU-U light device. This manuscript was supported by Dr. Jagdeo&#39;s residual funding. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

There was a significant increase in SCC-13 apoptosis following 30 min incubations of 0.5, 1, and 2 mM 5-ALA followed by 1,000 s of blue light. These results are consistent with our previously published research demonstrating that 5-ALA incubations of 30 min followed by blue light activation leads to a dose-dependent increase in the percentage of fibroblast cells undergoing apoptosis12,14.
This experimental approach to studying PDT has advantages and limitations. The described methods conform to clinical practice as a commercially available PS and light source were used. Researchers may customize the methods with different cell types, PSs, light sources, and irradiation parameters.However, there are limitations to this approach as this protocol is designed for adherent cells cultured in a monolayer. In clinical practice, differences in tissue architecture or disease pathology (i.e., hyperkeratosis or fibrosis) may decrease PS absorption or light penetration15. As a result, the treatment doses and experimental findings may not directly correspond to clinical practice. Spheroid tumor models and microfluidic chip have been studied as methods to more closely replicate tumor microenvironments16,17,18. Spheroids have inner and outer cellular niches that may differentially incorporate the photosensitizers and represent heterogeneous tumor architecture. Other researchers have used microfluidic chips to screen treatment conditions and vascular delivery of photosensitizers. However, microfluidic chips may be costly to develop or difficult to implement for researchers unfamiliar with the technique. Furthermore, spheroids and microfluidic chips may not reflect the clinical treatment of skin cancers in which photosensitizers are directly applied to the tumor surface without vascular dissemination. Researchers may need to evaluate the pros and cons of monolayer culture, spheroid tumor models, and microfluidic chips for studying PDT in different disease systems. As there are no immune cells or extracellular matrix, it is not possible to determine how PDT affects complex cell-cell interactions. Researchers may need to confirm in vitro experimental results using animal models and clinical trials. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments.
Temperature is known to alter the efficacy of PDT19.One study demonstrated that cell death, 5-ALA uptake, PP-IX formation, and cytokine release may be enhanced by incubating cells with 5-ALA at temperatures up to 44 &#176;C19. Incubation temperatures above 44 &#176;C may lead to thermal induced cell death, and incubation temperatures below 20 &#176;C may not lead to significant 5-ALA uptake and accumulation19. We recommend that researchers perform PS incubations on a temperature-adjustable heating block at a constant temperature to control for potentially confounding thermal effects. Additionally, it is important to incubate the photosensitizer for a sufficient amount of time to allow for the conversion of 5-ALA into PP-IX. In this protocol, SCC-13 cells were incubated with 5-ALA for 30 min, which successfully induced cell apoptosis. We have previously demonstrated that a 10 min incubation of 5-ALA did not significantly increase apoptosis in fibroblast compared to untreated control fibroblasts11,14. PP-IX starts accumulating in mouse skin after 5-ALA is incubating for six minutes at 37 &#176;C. Therefore, after ten minutes of 5-ALA incubation, there may not be sufficient accumulation of PP-IX to induce cell apoptosis20. We recommend a minimum incubation period of 20 to 30 min for 5-ALA based on our previous studies11,14. Researchers may need to optimize incubation period based on the laboratory setting, photosensitizer of interest, cell type, and clinical indication. Antibody titration and optimization experiments may be performed for best results using manufacturers guidelines. Other assays of interest may be performed following blue light photoactivation including dihydroethidium flow cytometric quantification of ROS.14
Variation in the amount of light delivered per unit of surface area (i.e., irradiance) and total irradiation dose (i.e., fluence) during the photoactivation phase may affect ROS formation and cell apoptosis21. The relationship between fluence, irradiance, and time can be described by the following equation:
Fluence (J/cm2) = Irradiance (W/cm2) x time (s)
As fluence is dependent on time, lengthening or shortening the photoactivation phases may change treatment efficacy. The irradiance is proportional to distance squared between the light source and the target tissue. As a result, if the light is too far away, there may not be sufficient light energy delivered to the targeted tissue to excite the PS. Alternatively, the irradiance may be increased by light reflecting off the surrounding surfaces. Therefore, we recommend performing light irradiations with the cell culture plates placed on a black surface to prevent light reflection. Researchers may acquire commercially available or FDA-approved blue light-emitting diodes and fluorescent devices to use in PDT experiments, but these devices may have different power outputs and light field uniformity. A wavelength-specific photometer should be used to measure the irradiance at the cell surface and uniformity of the light field before every experiment for consistent results. A commercially available diffuser may be used to enhance field uniformity, if necessary.
In summary, we have described an in vitro approach to investigating PDT by detailing theory, experimental methods, limitations, potential pitfalls, and recommendations for optimization. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration (FDA) has approved PDT for the treatment of actinic keratosis (AK), and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris1,2. Emerging non-dermatological uses for PDT include treatment of gastrointestinal, prostate, and gynecological cancers3. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scarring2.
In the first step of PDT, a PS is applied and allowed to accumulate intracellularly2. In the United States, topical 5-aminolevulinic acid (5-ALA) is commonly used to treat AKs. During the incubation phase, 5-ALA is converted into protoporphyrin IX (PP-IX) via the heme biosynthesis pathway in incorporated cells3. Cancer and pre-cancer cells have decreased ferrochelatase activity, which converts protoporphyrin IX (PP-IX), into the end product, heme. As a result, cancers cells accumulate PP-IX compared to normal cells4,5. This accumulation of PP-IX in cancer and pre-cancer cells relative to the surrounding tissue allows for PDT to be a targeted approach with minimal adverse events. Light irradiation leads to reactive oxygen species (ROS) generation when oxygen accepts excited electrons from PP-IX. PP-IX has a peak absorption in the ultraviolet spectrum with smaller peaks throughout the visible light spectrum, including blue and red light2. ROS formation may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover6,7,8,9,10.
PDT was developed in the 1970s and is an evolving therapeutic modality3. The FDA-approved protocol for the treatment of AKs recommends 5-ALA skin incubation for 14&#8211;18 h, but 1 to 2 h incubations are commonly used in clinical practice2. In vitro, we have shown that shorter 15 min 5-ALA incubations may increase fibroblast cell apoptosis compared to untreated fibroblasts11,12. Additionally, novel chlorin, phthalocyanine, and nanoparticle-based PSs are being studied to improve PDT efficacy3. Herein, we present an in vitro method to study PDT in an adherent squamous cell carcinoma cells. In this protocol, SCC-13 cells are incubated with 0, 0.5, 1.0, and 2 mM 5-ALA for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure is apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D (7-AAD) flow cytometry. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with other cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Preparation of additional control groups may be necessary, including unstained, single fluorophore stained, negative, and positive controls for flow cytometry. A comprehensive review of theory, protocols, experimental design, and gating for apoptosis/necrosis flow cytometry can be found elsewhere13.

<table border="0" cellpadding="0" cellspacing="0" width="1259">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Keratinocyte serum free medium&#160;</td>
			<td style="width:211px;">Thermofisher</td>
			<td style="width:204px;">17005042</td>
			<td style="width:623px;">Culture medium for SSC-13 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM low glucose glutamax</td>
			<td>Thermofisher</td>
			<td>10567022</td>
			<td>Possible culture medium for other cell types</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">6-well culture plates</td>
			<td>Thermofisher</td>
			<td>720083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Thermofisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA</td>
			<td>Thermofisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td>Thermofisher</td>
			<td>15250061</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLU-U light&#160;</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td>Other blue LED, flourescent, or halogen lights may used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-ALA</td>
			<td>DUSA</td>
			<td>Request from manufacturer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Atlanta Biologicals</td>
			<td>C17032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowCellect Annexin Red Kit</td>
			<td>Millipore Sigma&#160;</td>
			<td>FCCH100108&#160;</td>
			<td>Propidium iodide may replace 7-AAD. Comes with annexin-V, 7-AAD, and flow buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSCanto II</td>
			<td>BD</td>
			<td>Request from manufacturer</td>
			<td>Any flow cytometer may be used&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counting Chamber</td>
			<td>Hausser</td>
			<td>3120</td>
			<td>For cell counting and plating&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo</td>
			<td>FlowJo</td>
			<td>Request from manufacturer</td>
			<td>Flow cytometry analysis software</td>
		</tr>
	</tbody>
</table>

an in vitro approach to photodynamic therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

After SCC-13 cells were incubated for 30 min with 0, 0.5, 1, and 2 mM 5-ALA and irradiated with 1,000 s of blue light, there was a dose-dependent increase in cell apoptosis. Total apoptosis (mean &#177; standard error of mean) was 3.94 &#177; 0.34, 7.90 &#177; 0.52, 23.86 &#177; 0.52, and 38.33 &#177; 1.81 after 30 min of incubation with 0, 0.5, 1, and 2 mM 5-ALA, respectively, and 1000 seconds of blue light (Figure 1). We compared the mean percentage of apoptotic cell among 0, 0.5, 1, and 2 mM 5-ALA incubated groups using ANOVA. Cells treated with 1 and 2 mM 5-ALA had a significant increase in cell apoptosis compared to 0 mM 5-ALA treated cells. Figure 2A shows representative flow cytometric gating of forward scatter (FSC) versus side scatter (SSC) for SCC-13 cells incubated with 0, 0.5, 1, and 2 mM 5-ALA. The circle gating encloses the SCC-13 cell population. For this experiment, 7500 events were collected and the SCC-13 cell population gate captured at least 90% of events. Figure 2B shows a representative plot of the 7-AAD on the x-axis and annexin-V on the y-axis. The cell populations are gated into four quadrants (Q1 to Q4) to discriminate apoptotic cells. Q1 (high annexin-v, low 7-AAD) represents cells undergoing early apoptosis. Q2 (high annexin-v, high 7-AAD) represent cells undergoing late apoptosis or necrosis. Q3 (low annexin-v, high 7-AAD) represents cells undergoing necrosis. Q4 (low annexin-v, low 7-AAD) represents cells not undergoing apoptosis or necrosis. To calculate the percentage of cells undergoing apoptosis, we added the percentage of cells in Q1 and Q2. Inconsistency in 5-ALA incubation period length, incubation temperature, or photoactivation irradiation dose may alter PDT efficacy. Figure 3 demonstrates a representative annexin-V and 7-AAD flow cytometric plot when the incubation temperature is varied (i.e., 27, 36, or 42 &#176;C). There was a temperature dependent increase in apoptosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58190/58190fig1.jpg" /> 
Figure 1: Dose-dependent increase in apoptosis after thirty-minute incubation of 5-ALA. 5-ALA incubated at 36 &#176;C for thirty minutes followed by 1,000 s of blue light in SCC-13 cells. Bars represent average percent annexin-V positive cells in each treatment and control group. Experiments were performed in technical triplicate. Statistical significance was determined by ANOVA, with p &#60;0.05 denoted by an asterisk (*). Error bars represent the mean &#177; standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig1large.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/58190/58190fig2.jpg" /> 
Figure 2: Representative Flow Cytometry plots. (A) Representative forward scatter vs. side scatter cytometry plots in Arbitrary Units (AU) for x- and y-axis for 0, 0.5, 1, and 2 mM 5-ALA incubated cells. For this experiment, 7500 events were collected and the SCC-13 cell population gate captured 90% of events. (B) Representative annexin-V vs. 7-AAD flow cytometry plots in AU for x- and y-axis for 0, 0.5, 1, and 2 mM 5-ALA incubated cells. Q1: early apoptotic cells, Q2: late apoptotic/necrotic cells, Q3: necrotic cells, and Q4: viable cells. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58190/58190fig3.jpg" /> 
Figure 3: Incubating 5-ALA at different temperatures may alter PDT efficacy.&#160;Representative annexin-V vs. 7-AAD flow cytometry plots in AU for x- and y-axis for SCC-13 cells incubated with 0.5 mM 5-ALA at 27, 36, and 42 &#176;C. Q1: early apoptotic cells, Q2: late apoptotic/necrotic cells, Q3: necrotic cells, and Q4: viable cells. <a href="https://www.jove.com/files/ftp_upload/58190/58190fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

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8.6K Views.  University of California, Davis. Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Video: An In Vitro Approach to Photodynamic Therapy

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

A Multi-Modal Approach to Assessing Recovery in Youth Athletes Following Concussion

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can experience a range of short and long term neurobehavioral symptoms (somatic, cognitive and emotional/behavioral) that can have a significant impact on one&#8217;s participation in daily activities and pursuits of interest (e.g., school, sports, work, family/social life, etc.). Despite this, there remains a paucity in clinically driven research aimed specifically at exploring concussion within the youth sport population, and more specifically, multi-modal approaches to measuring recovery. This article provides an overview of a novel and multi-modal approach to measuring recovery amongst youth athletes following concussion. The presented approach involves the use of both pre-injury/baseline testing and post-injury/follow-up testing to assess performance across a wide variety of domains (post-concussion symptoms, cognition, balance, strength, agility/motor skills and resting state heart rate variability). The goal of this research is to gain a more objective and accurate understanding of recovery following concussion in youth athletes (ages 10-18 years). Findings from this research can help to inform the development and use of improved approaches to concussion management and rehabilitation specific to the youth sport community.

This article provides an overview of a multi-modal approach to assessing recovery following concussion in youth athletes. The described protocol uses pre- and post-concussion assessment of performance across a wide variety of domains and can inform the development of improved concussion rehabilitation protocols specific to the youth sport community.

Concussion is one of the most commonly reported injuries amongst children and youth involved in sport participation. Following a concussion, youth can ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...