Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00.000+00:00
Duration: 6 min 8 s
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (116159-02-2-WT011) and School of Life Sciences and Biotechnology for BK21 PLUS, Korea University.

1. Preparation of Medium, Compounds, and Reagents
<ol>
	<li>Prepare B16F10 growth complete medium. Supplement Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PEST).
	<ol>
		<li>To make 500 mL of complete medium, mix 445 mL of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) with 50 mL of FBS and 5 mL of PEST in a 1 L sterilized glass bottle. After mixing gently, filter the medium through a 0.2-&#181;m bottle-top filter to a new sterilized glass bottle and then store at 4&#160;&#176;C. 
		CAUTION: All cell culture techniques should be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility.</li>
	</ol>
	</li>
	<li>Prepare lysis buffer: 20 mM Tris (hydroxymethyl) aminomethane containing 0.1% Triton X-100 (v/v) buffer at pH 7.5 (pH adjusted with HCl). This buffer can be used for 3 months when stored at room temperature (RT). Add protease inhibitors to the lysis buffer right before use.</li>
	<li>Prepare tyrosinase inhibitor assay buffer. Prepare 1 M NaH2PO4 (monobasic) and 1 M Na2HPO4 (dibasic) stock solutions. Mix 46.3 mL of Na2HPO4 and 53.7 mL of NaH2PO4 to prepare a final volume of 0.1 M sodium phosphate buffer (pH 6.8).
	<ol>
		<li>Dilute the resulting mixture to 1 L (final volume) with H2O. Adjust the pH of the final solution to 6.8. This buffer can be used for 1 month when stored at 4&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Prepare tyrosinase substrate solution: 0.1% 3,4-dihydroxy-L-phenylalanine (DOPA, w/v) dissolved in tyrosinase inhibitor assay buffer (refer to Step 1.3). 
	CAUTION: Keep on ice while in use.</li>
	<li>Optionally, prepare 100 U/mL mushroom tyrosinase. Dissolve the lyophilized mushroom tyrosinase in tyrosinase inhibitor assay buffer. This solution can be used for 2 months when stored at &#8211;20&#160;&#176;C. The mushroom tyrosinase activity as well as cellular tyrosinase activity are calculated in the presence of cell materials. 
	CAUTION: The solution is aliquoted before freezing thus repeated freezing-and-thawing can be avoided. Keep the solution on ice while in use.</li>
	<li>Prepare 1 N NaOH solution. Weigh 4 g of NaOH in a volumetric flask and dissolve it in distilled water to make 100 mL.</li>
	<li>Prepare fixation solution (10% formalin). Dilute 27 mL of 37% formaldehyde with 73 mL of distilled water. Prepare fresh daily.</li>
	<li>Prepare ammoniacal silver stock solution. Prepare 5 mL of 10% silver nitrate solution in a volumetric flask. Add ammonium hydroxide solution dropwise, until the precipitate is completely dissolved. Add 200 &#956;L of silver nitrate solution (10%). The solution will become slightly cloudy. 
	CAUTION: Avoid contact and inhalation. Use a fume hood.</li>
	<li>Prepare ammoniacal silver working solution. Dilute 2.5 mL of ammoniacal silver stock solution with 7.5 mL of distilled water. Use once and then discard. 
	CAUTION: Avoid contact and inhalation. Use a fume hood.</li>
	<li>Prepare 0.1% gold chloride. Prepare 1 mL of 10% gold chloride and add 99 mL of distilled water in a volumetric flask. Prepare fresh daily.</li>
	<li>Prepare phosphate buffer saline (PBS). Add 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 in 800 mL of distilled water. Adjust the pH to 7.4 with HCl. Dilute the resulting mixture to 1 L (final volume) with distilled water.</li>
	<li>Prepare 5% (w/v) sodium thiosulfate solution dissolved in distilled water.</li>
</ol>
2. Cell Culture and Treatment
<ol>
	<li>Use commercially available B16F10 cells. Maintain the B16F10 cells in complete medium. Keep the cell culture flask in a humidified, 5% CO2 atmosphere incubator at 37&#160;&#176;C.</li>
	<li>Prepare test compounds, inhibitor positive control, and negative control samples.
	<ol>
		<li>Dissolve the test compounds in the appropriate solvents. Dissolve water soluble compounds in double-distilled water. Dissolve water insoluble compounds in appropriate organic solvent, e.g., DMSO.</li>
		<li>Here, use arbutin dissolved in double-distilled water (125 &#956;g/mL) as a positive control to assess the hypopigmentation activity compared with test compounds or a negative control. As a negative control (vehicle-treated control), use the solutions or buffers used in the test sample, e.g., double-distilled water, DMSO, ethanol.</li>
	</ol>
	</li>
	<li>Cell seeding and treatment
	<ol>
		<li>Seed B16F10 cells at 5 &#215; 104 cells/well in 24-well culture plates using complete medium and incubate in a CO2 incubator for 24 h. After incubation, aspirate the complete medium.</li>
		<li>Incubate cells with DMEM medium (without FBS and 1% penicillin/streptomycin) containing either test compounds, an inhibitor control, or a negative control in a CO2 incubator for 72 h. Check cells every 24 h&#160;under the microscope. After this step, go to Steps 3-5 for further experiments. 
		CAUTION: The mixture of the test substance and DMEM medium should be filtered through a 0.2 &#956;m filter.</li>
	</ol>
	</li>
</ol>
3. Measuring Cellular Tyrosinase Activity
<ol>
	<li>Using the method described in Step 2, remove media and rinse cells twice with 300 &#956;L of cold PBS.</li>
	<li>Place the cell culture plate on ice. Add 300 &#956;L of lysis buffer and incubate for 5 min. Then collect the cell lysate using a cell scraper and transfer the cell lysate to a 1.5-mL microcentrifuge tube.</li>
	<li>Homogenize the cell lysate with a cell homogenizer at 14,500 rpm for 2 s, 3 times on ice to obtain intracellular tyrosinase. Use the optimum condition specific for the homogenizer.</li>
	<li>Centrifuge for 10 min at 12,000 &#215; g at 4&#160;&#176;C and transfer the cell supernatant to a 1.5-mL microcentrifuge tube. Keep on ice while in use.</li>
	<li>Transfer 70 &#956;L of the supernatant to a 96-well clear polystyrene microplate.</li>
	<li>Add 140 &#956;L of a solution containing tyrosinase substrate (DOPA) to a 96-well clear polystyrene microplate, shake gently, and incubate for 2 h at 37&#160;&#176;C.</li>
	<li>Optional, add 70 &#956;L of 100 U/mL mushroom tyrosinase solution to each well and incubate for 2 h at 37&#160;&#176;C.</li>
	<li>Measure the tyrosinase activity at a wavelength of 475 nm using microplate reader.</li>
	<li>Normalize the tyrosinase activity to the protein concentration of each sample. Measure the protein concentration of each sample by a Bradford assay.</li>
</ol>
4. Measuring Melanin Content
<ol>
	<li>Using the method described in the Step 2, remove media and rinse cells twice with 300 &#956;L of cold PBS.&#160;</li>
	<li>Place the cell culture plate on ice. Add 300 &#956;L of lysis buffer and incubate for 5 min. Then collect the cell lysate using a cell scraper and transfer the cell lysate to a 1.5-mL microcentrifuge tube.</li>
	<li>Centrifuge for 10 min at 12,000 &#215; g at 4&#160;&#176;C.</li>
	<li>Transfer the supernatant to a new tube and measure total protein concentration for normalization. Measure the protein concentration of each sample by a Bradford assay.</li>
	<li>Add 300 &#956;L of 1 N NaOH to each pellet and incubate at 60&#160;&#176;C for 1 h.</li>
	<li>Centrifuge the dissolved solution for 10&#160;min at 12,000 &#215; g at 4&#160;&#176;C.</li>
	<li>Transfer 200 &#956;L of the supernatant to a 96-well microplate.</li>
	<li>Measure the melanin contents at a wavelength of 400 nm.</li>
	<li>Normalize the melanin contents to the protein concentration.</li>
</ol>
5. Fontana&#8211;Masson staining
<ol>
	<li>Fontana&#8211;Masson staining
	<ol>
		<li>Wash cells twice with 300 &#956;L of cold PBS.</li>
		<li>Add 200 &#956;L of 10% formalin to each well and incubate for 1 h at 4&#160;&#176;C.</li>
		<li>Rinse with distilled water.</li>
		<li>Add 200 &#956;L of pre-warmed ammoniacal silver working solution and incubate for 1 h at 37&#160;&#176;C or until the cells become yellow/brown in color. 
		CAUTION: Place freshly mixed ammoniacal silver working solution in a 58&#8211;60&#176;C water bath and allow adequate time for the temperature to equilibrate.</li>
		<li>Remove ammoniacal silver working solution and rinse&#160;with distilled water.</li>
		<li>Remove distilled water. Add 200 &#956;L of 0.1% gold chloride and incubate for 2 min at RT.</li>
		<li>Remove 0.1% gold chloride solution and rinse&#160;with distilled water.</li>
		<li>Remove distilled water. Add 200 &#956;L of sodium thiosulfate solution (5%, w/v) and incubate for 2 min.</li>
		<li>Remove sodium thiosulfate solution and rinse with distilled water.</li>
		<li>Remove distilled water. Add 200 &#956;L of Nuclear Fast Red and incubate for 5 min.</li>
		<li>Remove Nuclear Fast Red and rinse&#160;with tap water.</li>
		<li>Remove tap water and observe the stained cells under a microscope.</li>
	</ol>
	</li>
	<li>Fontana&#8211;Masson staining (Threshold Analysis Using ImageJ) 
	Note: An example of the image analysis procedure using ImageJ software is shown in the Supplemental Materials.
	<ol>
		<li>Open the program ImageJ.</li>
		<li>Select File | Open | Microscope Image.</li>
		<li>Select Image | Adjust | Color Threshold.</li>
		<li>To measure the area stained with Fontana&#8211;Masson, set the brightness parameter bar to zero and adjust the brightness bar to find the point at which all stained cells (black or dark brown) are included.</li>
		<li>Select Analyze | Analyze Particles. Check the summarize box in the analyze particles window and press OK. Obtain the summary result and paste into a spreadsheet. 
		Note: The description of the resulting box parameters are as follows: 
		Slice: Name of each image 
		Count: Number of stained cells 
		Total Area: Total area of the counted cells 
		Average Size: Total Area/Count 
		% Area: Stained Area/Total Area &#215; 100%; total area is calculated automatically.</li>
		<li>Calculate the relative area stained with melanin using the value of Total Area. 
		Relative melanin stained area (%) = Value of total cellular area of test sample/average total area of control &#215; 100, i.e.,</li>
	</ol>
	</li>
</ol>

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The authors have no disclosures to report.

We presented protocols for evaluating the hypopigmentation activity of test compounds using cultured melanocytes. The representative results showed the hypopigmentation effect of arbutin, a tyrosinase inhibitor that inhibited tyrosinase activity and cellular melanin content. These methods are widely used in anti-melanogenic activity research. Using these assays, we have also successfully identified several bioactive compounds that have melanogenesis inhibitory effects on B16F10 cells in the past decade9...

Melanin plays a critical role in the physiology, pathology, and toxicology of several organs including the skin, eyes, and brain1. Major functions of melanin are photo-screening and biochemical effects. Melanin absorbs near-infrared and visible light as well as ultraviolet (UV) radiation, with increased absorption rates at shorter wavelengths of light; thus, melanin protects tissues from damage caused by visible light or UV radiation2. Melanin is an antioxidant and has an affinity for metals and other toxic chemicals; therefore, it can protect tissues from oxidative and chemical stress3. However, the excessive production of melanin causes dermatological issues.
The quantity and quality of melanin in the skin and iris are the most important determinants of the color of the iris and skin. Individuals may have different skin color preferences; some favor tanned skin, while others favor lighter skin colors. Depending on these consumer profiles, hypopigmentation cosmetics have been developed to satisfy individual markets for favored skin colors4. Accordingly, studies of hypopigmentation and anti-melanogenic activity are important both scientifically and practically.
Melanogenesis is the complex process of melanin biosynthesis through a series of enzymatic and spontaneous chemical reactions in melanocytes. One melanocyte is surrounded by approximately 36 keratinocytes and melanocytes are the melanin synthesis factories that distribute their product to neighboring keratinocytes. In skin, the melanin produced and stored in the melanosomal compartment of melanocytes is transported to neighboring keratinocytes in the epidermis via dendrites.
L-Tyrosine serves as the initial substrate for melanogenesis and the enzyme tyrosinase catalyzes two consecutive reactions that convert L-tyrosine into 3,4-dihydroxyphenylalanine (DOPA) and then into dopaquinone. These reactions are the rate-limiting step in melanogenesis5,6. Accordingly, hypopigmentation activity can first be measured by assessing cellular tyrosinase activity directly. To do so, melanocyte extracts containing tyrosinase are incubated with DOPA and the dopaquinone produced in the samples can be measured by spectrophotometry at 475 nm. The values are normalized by the protein concentrations of the samples, and substances with hypopigmentation activity result in less dopaquinone formation compared with controls.
Secondly, hypopigmentation activity can be quantified by measuring melanin in cultured melanocytes directly. After treating cells with the test material, melanin is extracted under alkaline conditions and the melanin content is quantified by spectrophotometry at 400 nm. A hypopigmentation agent will result in a lower melanin content than the controls7.
Finally, the hypopigmentation activity can be quantified by Fontana-Masson melanin staining and subsequent image analysis. In Fontana-Masson staining, melanin granules reduce ammonia-silver nitrate to a visible black metallic state and the black areas of cells in microscopic images represent the amount of melanin.
A hypopigmentation agent usually gives consistent and comparable results with these three methods, which confirms that the activity of the substance is valid. Alternatively, it may be useful to measure the expression of key genes and proteins in melanogenesis in response to a test substance to examine hypopigmentation activity. In addition to tyrosinase, tyrosinase-related proteins (TRP-1) and dopachrome tautomerase (TRP-2) are critical enzymes in melanogenesis8. The transcription factor microphthalmia-associated transcription factor (MITF) is a master regulator in melanogenesis and quantification of its gene/protein expression levels or a promoter activity assay can also be used to assess hypopigmentation activity.

<table><tbody><tr><td>3,4-Dihydroxy-l-phenylalanine</td><td>Sigma</td><td>D9628</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma</td><td>#320145</td><td></td></tr><tr><td>Arbutin</td><td>Fluka</td><td>#10960</td><td></td></tr><tr><td>DMEM</td><td>HyClone</td><td>SH30243.01</td><td></td></tr><tr><td>FBS</td><td>HyClone</td><td>SH30084.03</td><td></td></tr><tr><td>Formalin</td><td>Yakuri Pure Chemicals</td><td>#16223</td><td>37%</td></tr><tr><td>Gold chloride</td><td>American MasterTech</td><td>AHG0226</td><td>0.10%</td></tr><tr><td>HCl</td><td>Samchun chemical</td><td>H0255</td><td></td></tr><tr><td>KCl</td><td>Bio basic Canada Inc.</td><td>PB0440</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>#60218</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO4</td><td>J.T.Baker</td><td>#3817-01</td><td></td></tr><tr><td>NaCl</td><td>Duksan pure chemicals</td><td>#81</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>655104</td><td></td></tr><tr><td>Nuclear fast red</td><td>Merck</td><td>100121</td><td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td></tr><tr><td>Penicillin and streptomycin solution</td><td>HyClone</td><td>SV30010</td><td></td></tr><tr><td>Silver nitrate</td><td>Duksan Pure Chemicals</td><td>#900</td><td></td></tr><tr><td>Sodium phosphate dibasic (Na2HPO4)</td><td>J.T. Baker</td><td>#3817-01</td><td></td></tr><tr><td>Sodium phosphate monobasic (Na2HPO4)</td><td>Sigma</td><td>S5011</td><td></td></tr><tr><td>Sodium thiosulfate pentahydrate</td><td>Duksan Pure Chemicals</td><td>#2163</td><td></td></tr><tr><td>Tris(hydroxymethyl)aminomethane</td><td>Bio Basic Canada</td><td>TB0196</td><td></td></tr><tr><td>Triton X-100</td><td>Union Carbide</td><td>T8787</td><td></td></tr><tr><td>Tyrosinase from mushroom</td><td>Sigma</td><td>T3824</td><td>25 KU</td></tr></tbody></table>

quantification of hypopigmentation activity in vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

Representative results for the hypopigmentation activity of arbutin, an anti-melanogenic compound, in B16F10 melanocytes are shown below. Figure 1A shows that arbutin significantly suppressed the cellular tyrosinase activity compared with the vehicle-treated control. Similarly, the melanin content of cells stimulated with arbutin was significantly reduced compared with the controls (Figure 1B). Microscopic images of cells stained...

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Quantification of Hypopigmentation Activity In Vitro

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

11.2K Views.  Korea University. We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

Video: Quantification of Hypopigmentation Activity In Vitro

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Medicine

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic chemotherapy due to their precise spatiotemporal activation by directing a benign, non-ionizable light to a specific location within the patient&#39;s body. This paper presents a set of methods to evaluate the in vitro potency and ex vivo efficiency of the photoactivation of photocontrolled, biologically active compounds as well as the in vivo efficacy at early stages of drug development. The methodology is applied to anticancer cytotoxic peptides, namely, the diarylethene-containing analogs of a known antibiotic, gramicidin S. The experiments are performed using 2D (adherent cells) and 3D (spheroids) cell cultures of a cancer cell line (Lewis lung carcinoma, LLC), live tissue surrogates (pork meat mince), and an allograft cancer model (subcutaneous LLC) in immunocompetent mice. The selection of the most effective compounds and estimation of realistic phototherapeutic windows are performed via automated fluorescence microscopy. The photoactivation efficiency at varying illumination regimens is determined at different depths in a model tissue, and the optimal light dosage is applied in the final therapeutic in vivo experiment.

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

This protocol presents a set of experiments adopted for the evaluation of photoswitchable anticancer peptides, that can be used in the preclinical screening of such compounds. This includes cytotoxicity assessment in 2D and 3D cell cultures, the evaluation of ex vivo (model tissue) photoisomerization efficiency, and in vivo efficacy.

Photocontrolled, biologically active compounds are an emerging class of &#34;smart&#34; drug candidates. They provide additional safety in systemic ...

Chemistry

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, influenza, diphtheria, and viral hemorrhagic fevers. We have demonstrated that plasmid-DNA (pDNA) vaccine delivery into the skin elicits robust humoral responses in the guinea pig. However, the use of this animal to model immune responses was somewhat limited in the past due to the lack of available reagents and protocols to study T cell responses. T cells play a pivotal role in both immunoprophylactic and immunotherapeutic mechanisms. Understanding T cell responses is crucial for the development of infectious disease and oncology vaccines and accommodating delivery devices. Here we describe an interferon-gamma (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay for guinea pig peripheral blood mononuclear cells (PBMCs). The assay enables researchers to characterize vaccine-specific T-cell responses in this important rodent model. The ability to assay cells isolated from the peripheral blood provides the opportunity to track immunogenicity in individual animals.

The development of the interferon-gamma ELISpot assay for guinea pig PBMCs allows the characterization of T-cell responses in this highly relevant model for studying infectious diseases. We have applied the assay to measure T cell responses associated with intradermal delivery of DNA vaccines.

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...

Immunology and Infection

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.

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.

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

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Biology

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Quantifying Retinal Pigment Epithelial Phagocytosis and Lipofuscin Accumulation

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.

Author Spotlight: Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures  

This protocol describes a lipofuscin accumulation model in highly differentiated and polarized human retinal pigment epithelial (RPE) cultures and an improved outer segment (OS) phagocytosis assay to detect the total OS consumption/degradation capacity of the RPE. These methods overcome the limitations of previous lipofuscin models and classical pulse-chase outer segment phagocytosis assays.

The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging ...

Modeling and Analyzing Retinal Degeneration

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

Author Spotlight: Unraveling the Pathogenesis of Age-Related Macular Degeneration and Discovering Potential Therapies 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://www.jove.com/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Quantification of Hypopigmentation Activity In Vitro

Summary

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Chapters in this video

Biochemical Measurement of Neonatal Hypoxia

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

In Vitro and In Vivo Evaluation of Photocontrolled Biologically Active Compounds - Potential Drug Candidates for Cancer Photopharmacology

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

An In Vitro Approach to Photodynamic Therapy

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

Improved Lipofuscin Models and Quantification of Outer Segment Phagocytosis Capacity in Highly Polarized Human Retinal Pigment Epithelial Cultures

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells