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 decade9,10,11,12,13,14.
Melanin is produced by melanosomes in various pigment cells, including melanocytes in skin. In this study, we used B16F10 melanoma cells that produce melanin, which causes the culture medium to become dark brown or black15. Primary melanocytes might be biologically important tools for studying in vivo effects, but due to their limited proliferative capacity and the variability in phenotypes generated, cultured melanocytes are considered a reliable in vitro model16. Melanogenesis of this cell line is comparable with those of primary melanocytes. This method better represents the cellular environment than the mushroom tyrosinase activity assay, in which the enzyme is directly incubated with the substrate and hypopigmentation test compound.
Before performing these experiments, it might be necessary to perform the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay or comparable assays, such as the cell violet assay, with the test compound(s). If a test substance affects cell proliferation or exhibits cytotoxicity, then melanocyte growth could be affected, and the results might not correctly represent the cellular tyrosinase activity. Chemicals or natural substances often have some cytotoxicity at high concentrations and this is particularly important for substances with mild activity. It is also not uncommon for substances and natural extracts to promote cell proliferation and differentiation. Consequently, the potential for additional bioactivity causing cell growth should be examined, although these effects on cell growth could be adjusted by normalizing the activity with the protein concentration.
Various mechanisms could result in hypopigmentation independent of tyrosinase activity. Melanogenesis is a complex biosynthetic pathway that involves multiple signal transduction pathways and transcription factor activities17. Therefore, the expression of key genes and proteins and melanogenic signal transduction pathways could be examined for an in-depth study of hypopigmentation. MITF is an important transcription factor in melanogenesis and &#945;-melanocyte-stimulating hormone (&#945;-MSH) can stimulate melanogenic signal transduction pathways18,19. Depending on their known biological characteristics, related signal transduction pathways could be studied further.
When measuring melanin content, the obtained values should also be normalized by the protein concentration, so that the results correctly represent the melanin content per cell or per culture area20. Additionally, phenol-red-free medium is recommended in the compound treatment step to avoid the negative effects of phenol red on absorbance.
For melanin staining, we used the Fontana-Masson stain, which detects argentaffin substances, such as melanin and argentaffin cells21. This technique is also used for paraffin or frozen section samples following the deparaffinization step. The reagents used in this experiment are harmful; contact and inhalation should be avoided, and the reagents should be used only in a fume hood whenever possible. After staining, ImageJ is used to calculate the stained area. With Fontana-Masson staining, it can be difficult to interpret faint staining in sparsely positive cells; however, this may not be an issue for melanin-rich melanocytes. The silver nitrate, ammoniacal silver, sodium thiosulfate, and ammonium hydroxide used in the experiments should be handled with care due to their toxicity.
These are widely used are methods in the field of melanogenesis and hypopigmentation because they are rapid, handy, cost-effective, and reliable. These methods may be useful for investigators who want to identify novel compounds or extracts with anti-melanogenic or pro-melanogenic activities. They could also be used to assess melanocyte cell growth or cellular activity. One limitation is the reproducibility of the results on human skin, which is a complex organ in which homeostasis is maintained in response to various environmental, chemical, and biological factors. Therefore, hypopigmentation agents confirmed by these methods in vitro might not have significant effects on human skin in vivo.
In summary, these protocols are an important initial step in screening test compounds for their inhibitory effects on melanogenesis. We suggest that these methods can explain not only the inhibitory effects on melanogenesis but also the mechanism involved. After bioactive candidate compounds are identified, further testing may lead to studies of the biological mechanisms or commercial applications, followed by in vivo use.

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 with Fontana-Masson stain are shown in Figure 1C. Arbutin treatment decreased the area of black pigment compared with the control.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58185/58185fig1.jpg" /> 
Figure 1: Arbutin suppressed melanogenesis in melanocytes. B16F10 cells were treated with arbutin (125 &#956;g/mL) for 72 h. A. Cellular tyrosinase activity. B. Melanin content. C. Melanin was visualized with Fontana-Masson stain. Data are the means &#177; SEM (n = 4). Statistical comparisons were performed using Student&#39;s t-test (p values&#60;0.05 were considered to be statistically significant). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58185/58185fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.3K Views.  Korea University. Deze methode kan nuttig zijn bij de ontwikkeling van functionele cosmetica en huidagent met anti-melanogene activiteiten. Het is er om uit te voeren en het resultaat kan snel worden verkregen. Bovendien kan deze methode nuttig zijn voor onderzoekers, die de effecten of de verbindingen willen identificeren of onderzoeken of de antimelanogene of promelanogene activiteiten willen onderzoeken.Om te meten tyrosinase activiteit beginnen met het zaaien van B16-F10 Melaniciden op een vijf keer...

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

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Mouse models can be useful tools for investigating the biology of the retinal pigmented epithelium (RPE). It has been established that mice can develop an array of RPE pathologies. Here, we describe a phenotyping protocol to elucidate and quantify RPE pathologies in mice using light, transmission electron, and confocal microscopy.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal ...

Genetics

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

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 ...

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 ...

Biology

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a common approach relies on plating cells treated with different concentrations of antifungal molecules on agar plates and then counting the colonies to determine the relationship between molecule concentration and growth inhibition. This method requires many plates and substantial time to count the colonies. Another common approach eliminates the plates and counting of colonies by visually inspecting cultures treated with antifungal agents to identify the minimum concentration required to inhibit growth; however, visual inspection produces only qualitative results, and information on growth at subinhibitory concentrations is lost. This protocol describes a method for measuring the susceptibility of C. albicans to antifungal peptides. By relying on optical density measurements of cultures, the method reduces the time and materials needed to obtain quantitative results on culture growth at different peptide concentrations. The incubation of the fungus with peptides is performed in a 96-well plate using an appropriate buffer, with controls representing no growth inhibition and complete growth inhibition. Following the incubation with the peptide, the resulting cell suspensions are diluted to reduce peptide activity and then grown overnight. After overnight growth, the optical density of each well is measured and compared to the positive and negative controls to calculate the resulting growth inhibition at each peptide concentration. The results using this assay are comparable to the results using the traditional method of plating the cultures on agar plates, but this protocol reduces plastic waste and the time spent on counting colonies. Although the applications of this protocol have focused on antifungal peptides, the method will also be applicable to testing other molecules with known or suspected antifungal activity.

Quantifying the Antifungal Activity of Peptides Against Candida albicans

This protocol describes a method for obtaining quantitative data on the antifungal activity of peptides and other compounds, such as small-molecule antifungal agents, against Candida albicans. Its use of optical density rather than counting colony-forming units to quantify growth inhibition saves time and resources.

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

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.remotexs.ntu.edu.sg/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.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.&#160;J. Vis. Exp.&#160;(2016).
...

JoVE Encyclopedia of Experiments

Cancer Research

Lung Cancer

In vitro study

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

Quantification of Hypopigmentation Activity In Vitro

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