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 method may be helpful in development of functional cosmetics and skin agent with anti-melanogenic activities. It's there is to perform and the result can be obtained quickly. In addition, this method may be useful for researchers, who want to identify or investigate the effects or the compounds or investigate the antimelanogenic or promelanogenic activities.

To measure to Tyrosinase activity begin by seeding B16-F10 Melanicides at a five times 10 to the fourth cells per well of a 24 well plate concentration in complete medium for 24 hours in a cell culture incubator. The next day, replace the supernatant in each well with DMEM supplemented with freshly prepared test compound and inhibitor control or a negative control and return the plate to the incubator for another 72 hours. At the end of the treatment rinse the wells two times with 300 microliters of cold pbs per well and place the plate on ice.

Next add 300 microliters of lysis buffer to each well using a cell scraper to collect the cell lysates after five minutes. Transfer the resulting cell particle suspensions to individual 1.5 milliliter micro-centrifuge tubes. And homogenize the lysates three times for two seconds at 14, 500 rpm on ice to release the intercellular Tyrosinase.

Pellet the cell debris by centrifugation and transfer the supernatants to individual 1.5 milliliter centrifuge tubes on ice. Allocate 70 microliters of each supernatant to individual wells of a clear 96 well polystyrene micro plate and add 140 microliters of Tyrosinase substrate solution to each well of supernatant. Shaking the plate gentle to mix the well contents.

Then incubate the plate at 37 degrees celsius for two hours. And measure to Tyrosinase activity at 475 nanometers on a microplate reader. To measure the melanin content of the cells after collecting the lysate of the treated cells as demonstrated.

Collect the lysates by centrifugation and transfer the supernatants to new tubes. Add 300 microliters of one normal sodium hydroxide to each pellet for a one hour incubation at 60 degrees Celsius. Followed by centrifugation of the dissolved cell suspensions.

Then transfer 200 microliters of the supernatants to each well of a 96 well plate. And measure the melanin contents on a microplate reader at a 400 nanometer wavelength. For Fontana-Masson Staining, wash the treated cells two times with 300 microliters of cold pbs and fix the cells with 200 microliters of 10%formalin for one hour at four degrees Celsius.

Rinse the thick cells with distilled water and add 200 microliters of 58 to 68 degrees celsius Ammoniacal silver working solution to each well. For one hour incubation at 37 degrees Celsius or until the cells become yellow-brown in color. Rinse the stained cells with distilled water followed by two minute incubation in 0.1%gold chloride at room temperature.

Rinse the gold chloride treated cells with distilled water and add 200 microliters of sodium thiosulfate solution to the cells. After two minutes rinse the cells again. And label the cells with 200 microliters of nuclear fast red per well for five minutes.

Then wash the stained cells with tap water before imaging under a light microscope. For threshold analysis, open the images in image J and select image, adjust, and Color Threshold. To measure the area stained with Fontana Masson set the brightness parameter bar to zero and adjust the brightness to the point at which all of the black or brown stained cells are included.

Select Analyze and Analyze Particles and check the summarize box in the Analyze Particles window then click okay to obtain a summary of the results and copy and paste the data into a spreadsheet. Arbutin treatment significantly suppresses cellular Tyrosinase activity compared to control vehicle treated cells. Similarly the melanin content of cells stimulated with Arbutin is significantly reduced compared to the controls.

Although the cells are morphologically similar between treatment groups, Arbutin decreases the area of black pigment compared to the control treated cells. This method are useful for activities screening using our compound library. There are over hundreds of samples to be tested quickly.

Moreover this method can also be used to investigate the mechanism involving melanogenesis. After the bioactive candidates compounds have been identified, for their testimony to the study of biological mechanisms or commercial applications.