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>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</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

11.1K 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

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...