CLIAMS:1) An in-vitro assay for testing a compound as a promoter or inhibitor for a biomolecule transfer between cell(s), said assay comprising steps:
a. treating said test compound with cultured cell(s) for 20 to 24 hours, said cell(s) being pre-cultured and seeded into surface modified welled microtiter plates, wherein the confluency level of said cell(s) during treatment with said test compound is greater than 80%;
b. treating the cells resulting from step (a) with a carboxylated modified fluorescent microsphere(s) for 4 to 24 hours followed by incubation for the another period of 4 to 24 hours wherein said microsphere being suspended in a medium free from calcium and magnesium ions;
c. multiple washing the cells resulting from step (b) with a buffer solution free from calcium and magnesium ions; and
d. quantifying the fluorescence signal of cell(s) resulting from step (c) by a fluorescence signal quantifier to determine total uptake of said biomolecule by said cell for determining said test compound as the inhibitor or promoter of the melanin transfer.
2) The assay as claimed in claim 1, wherein said biomolecule is melanin.
3) The assay as claimed in claim 1, wherein the presence of fluorescence signal indicates the test compound as the promoter of the melanin transfer between cells.
4) The assay as claimed in claim 1, wherein the absence of fluorescence signal indicates the test compound as the inhibitor of the melanin transfer between cells.
5) The assay as claimed in claim 1, wherein said assay performs the assay in 6 to 7 hours per 96 samples.
6) The assay as claimed in claim 1, wherein said cell is a melanocyte and/or keratinocyte.
7) The assay as claimed in claim 1, wherein said modified fluorescent microsphere(s) has size of less than 0.5 micron.
8) The assay as claimed in claim 7, wherein said modified fluorescent microsphere(s) has size of 0.2 micron.
9) The assay as claimed in claim 1, wherein said modified fluorescent microsphere(s) is a polystyrene bead.
10) The assay as claimed in claim 1, wherein said buffer has a pH ranging from 7.1 to 7.5.
11) The assay as claimed in claim 10, wherein said buffer solution is a phosphate buffered saline.
12) The assay as claimed in claim 1, wherein said medium is Dulbecco’s Modified Eagle’s Medium.
13) The assay as claimed in claim 1, wherein said fluorescence signal quantifier is a microplate reader.
14) A kit for testing a compound as a promoter or inhibitor of a biomolecule transfer between cell(s)through the assay as claimed in claim 1, said kit essentially comprising:
a) cell(s) cultured in a carboxylated modified microtiter plate;
b) a test compound
c) a carboxylated modified fluorescent microsphere(s) wherein said microsphere(s) being suspended in a medium free from calcium and magnesium ions;
d) a buffer solution, wherein said buffer is free from calcium and magnesium ions;
e) a fluorescence signal quantifier.
15) The kit as claimed in claim 14, wherein said biomolecule is melanin.
16) The kit as claimed in claim 14, wherein the presence of fluorescence signal indicates the test compound as the promoter of the melanin transfer between cells.
17) The kit as claimed in claim 14, wherein the absence of fluorescence signal indicates the test compound as the inhibitor of the melanin transfer between cells.
18) The kit as claimed in claim 14, wherein said assay is efficient to perform the complete test in 6 to 7 hours per 96 samples.
19) The kit as claimed in claim 14, wherein said cell is a melanocyte and/or keratinocyte.
20) The kit as claimed in claim 14, wherein said modified fluorescent microsphere(s) has size of less than 0.5 micron.
21) The kit as claimed in claim 20, wherein said modified fluorescent microsphere(s) has size of 0.2 micron.
22) The kit as claimed in claim 14, wherein said modified fluorescent microsphere(s) is a polystyrene bead.
23) The kit as claimed in claim 14, wherein said buffer has a pH ranging from 7.1 to 7.5.
24) The kit as claimed in claim 23, wherein said buffer solution is a phosphate buffered saline.
25) The kit as claimed in claim 14, wherein said medium is Dulbecco’s Modified Eagle’s Medium.
26) The kit as claimed in claim 14, wherein said fluorescence signal quantifier is a microplate reader.
,TagSPECI:Field of the Invention
The present invention relates generally to a high throughput in-vitro assay for testing a compound for its tendency to promote or inhibit the melanin transfer between cell(s). The in assay/method also allows testing the extent of a biomolecule uptake(n) or transfer(ed), preferably melanin, in a cell or from a cell.
The invention further provides a kit for performing the said in-vitro assay.
Background and the prior art
Melanocytes are neural crest-derived cells distributed along the basal layer of the epidermis and hair bulb. These cells synthesize melanin pigment within unique melanocyte-specific organelles called melanosomes. Mammalian skin and hair/wool/fur colour is determined by melanin quantity and quality (brown/black eumelanin or red/yellow pheomelanin) in a process that involves a large number of steps regulated at multiple control points by a range of different molecules and compounds.
A number of assay formats are currently available for evaluation of test compound or biomolecule(s) to act as melanin transfer triggering agent/inhibiting agent. There are reports for various assay formats for evaluation of biomolecule uptake or transfer between the cells. One of the method involves evaluation by quantitative phagocytosis assay wherein the cells are seeded in a 96-well microtiter plate and treated with triggers/inhibitors/test compounds for 48 hours followed by rinsing with serum-containing medium and cultured for an additional 1 hour. Following each treatment, cells are incubated for 4 hours with labeled (eg. Fluorescent labeled)bioparticles (eg. E. coli K-12). The bioparticle suspension is aspirated, and Trypan Blue is added for 1 minute to quench extracellular fluorescence and then measured at suitable wavelength using a microplate reader. However, this method has various shortcomings like high background noise and so low signal to noise ratio, high standard deviations introduced and low reproducibility, failure to evaluate phagocytosis/melanin transfer/uptake between controls and test compounds and therefore no validation could be made and the test thereby gives false positives, which is as illustrated in Figure 1. In figure 1, the red color indicates cell membranes (boundaries) stained with Cell Mask membrane dye while green indicates Fluorescent labeled bioparticles (eg. E. coli K-12) which shows that the bioparticles were found outside the cells and stuck on the plate giving noise and false signal.

Another method for assaying Melanin transfer by the test agents is a co-culture method wherein the primary melanocytes are cultured with keratinocytes in chambered slides/coverslips in 24-well or 12 well plates, followed by treatment with triggers/inhibitors/test compounds for desired time. The transfer of melanosomes/melanin granules from melanocytes to keratinocytes is tracked by labeling them, followed by either microscopic imaging or flow cytometry. However this method is very tedious (as it is difficult to culture 2 cell types together), time-consuming and provides low throughput.
Yet another method known in the literature is the use of fluorescent polystyrene beads/microspheres surrogates for melanin uptake studies and for testing a compound as the melanin transferring agent or inhibiting agent, wherein the beads are either counted or fluorescence intensity quantified by imaging under microscope. However even this method is low-throughput and yields a poor signal to noise ratio (high background noise) therefore can result in false positives and false negatives. As illustrated in Figure 2, the counting of positive signals becomes very difficult owing to clustering of beads. Also measurement of intensity by microscope aided software is also not possible by employing this method.The green color in figure 2 indicates cell membranes (boundaries) stained with Wheat Germ Agluttinin (WGA) while the red color indicates fluorescent beads. As evident in Figure 2, the beads largely remained outside the cells, stuck on the culture plate and also on the cells forming clusters. This poses a problem of high background noise even if fluorescence intensity is quantified.
WO2010029115 A1 discloses a method useful in studying or modulating skin or hair pigmentation. The document discloses testing the ability of a substance to modulate the transfer of melanin from a melanocyte to a keratinocyte.The document also discloses use of plant extracts in compositions as well as to method comprising the topical use of such compositions for reducing or enhancing the skin or hair pigmentation. The plant extract comprises the extract of soybean, an extract of Artocarpus genus plant, an extract of Cyathea genus plant, an extract of Secale genus plant, an extract of Thalassiosira genus plant, an extract of Buddleja genus plant etc. for mediating the transfer of melanin between cells. The patent application essentially relates to testing of a substance as melanin regulator in terms of inhibitor/promoter for transferring the melanin between cells by analyzing the reaction of melanocyte and/or keratinocyte in the presence of that substance.
A non-patent literature titled “The silver locus product (Silv/gp100/Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture” (ExpDermatol. 2008 May;17(5):418-26. doi: 10.1111/j.1600-0625.2008.00702.x. Epub 2008 Mar 6.) by Singh SK, Nizard C, Kurfurst R, Bonte F, Schnebert S, Tobin DJ discloses in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, determination of melanin using gp100 as tracker is disclosed. Transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. Further, the melanosome transfer to keratinocytes has been detected using gp100 in matched melanocyte–keratinocyte co-culture. This technology disclosed in the document uses gp100 (protein) positive-melanosome co-culture assay for the detection of melanocyte fragment phagocytosis by keratinocytes as well as the detection of melanosomes within melanocytefilopodia. The authors have used a co-culture (keratinocytes and melanocytes) method to quantify the melanosome transfer by wherein the two cells are cultured together for 72hrs, followed by cell fixation, an overnight immunostaining with antibody, microscopic imaging and counting/scoring fluorescent spots individually, per cell (within keratinocytes) in five random microscopic fields per well, under a confocal microscope. This technology since based on the co-culture method suffers with the difficulties of the tedious co-culturing system (using 2 cell types), quantification involves cell fixation with methanol, immunostaining overnight, imaging and manually counting gp 100-positive spots per well/sample; and therefore is a low throughput technique.
Another non-patent literature titled “Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system” (Pigment Cell Res. 2001 Jun;14(3):185-94, written by Minwalla L, Zhao Y, Cornelius J, Babcock GF, Wickett RR, Le Poole IC, Boissy RE discloses use of Confocal fluorescence and electron microscopic examination to study the transfer ability of melanosomes from melanocytes to keratinocytes by labeling melanosme with gold dextrin or melanocytes with green fluorescence dye. However, these methods are complicated, time consuming, and ineffective for the quantitative analysis of melanosome transfer.

Flow cytometry can also be used to study melanosome transfer. In particular, flow cytometry has been applied by double-labeling with fluorescein isothiocyanate (FITC)-conjugated antibody against the melanosomal-associated protein TRP-1, and with Cys5-conjugated antibody against the keratinocyte-specific marker keratin, known through non-patent literature by Lin HC, Shieh BH, Lu MH, Chen JY, Chang LT, Chao CF “A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro” (Pigment Cell Melanoma Res. 2008;21:559–564). However, these methods were only applied to malignant cells and quantitative analysis was not easy.
Therefore, there is a need for an efficient, high throughput and a noiseless in-vitro assay for testing a compound as a promoter or inhibitor of the transfer of a biomolecule, preferably melanin, between cell(s).
Objects of the Invention
An object of the present invention is to overcome the drawback / disadvantages of the present invention.
Yet another object of the present invention is to provide an in-vitro assay for testing a compound for its tendency of triggering or inhibiting a biomolecule transfer in a cell.
Yet another object of the present invention is to provide a high throughput in-vitro assay which can test a compound for its tendency of triggering/inhibiting melanin transfer in a cell.
Yet another object of the present invention is to provide a high throughput in-vitro assay which can test the quantity of the melanin uptake/transfer(ed) in a cell.
Yet another object of the present invention is to provide a kit which can test a compound for its tendency of triggering/inhibiting the melanin transfer in a cell.
Yet another object of the present invention is to provide a kit for testing the quantity of melanin uptake/transfer(ed) in a cell.
Yet another object of the present invention is to provide a minimum noise in-vitro assay for testing a compound as melanin transfer agent or inhibitor agent.
Yet another object of the present invention is to provide a minimum noise in-vitro assay for determining the quantity of the uptake/ transfer of melanin in a cell.
Yet another object of the present invention is to provide an in-vitro assay which allows testing of 96 samples/6 to 7 hours for assessing their role as melanin transfer inhibitor or a promoter agent.
Yet another object of the present invention is to provide an in-vitro assay wherein the quantification of melanin transfer can be done using a microplate reader.
Yet another object of the present invention is to provide a kit wherein the quantification of melanin transfer can be done using a microplate reader.
Summary of the Invention
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.
In an aspect of the Invention, there is provided an in-vitro assay for testing a compound as a promoter or inhibitor of melanin transfer between cell(s), said assay comprising steps:
a. treating said test compound with cultured cell(s) for 20 to 24 hours, said cell being pre-cultured and seeded into surface modified welled microtiter plates, wherein the confluency level of said cell during treatment with said test compound is greater than 80%
b. treating the cells resulting from step (a) with a carboxylated modified fluorescent microsphere(s) for 4 to 24 hours followed by incubation for the another period of 4 to 24 hours wherein said microsphere being suspended in a medium free from calcium and magnesium ions;
c. multiple washing the cells resulting from step (b) with a buffer solution free from calcium and magnesium ions;
d. Quantifying the fluorescence signal of cell(s) resulting from step (c) by a fluorescence signal quantifier to determine total uptake of said biomolecule by said cell for determining said test compound as the inhibitor or promoter of the melanin transfer
In another aspect of the Invention, there is provided a kit for testing a compound as a promoter or inhibitor of melanin transfer between cell(s) through the assay as mentioned above, said kit essentially comprising:

a) cell(s) cultured in a carboxylated modified microtiter plate;
b) a test compound
c) a carboxylated modified fluorescent microsphere(s) wherein said microsphere(s) being suspended in a medium free from calcium and magnesium ions;
d) a buffer solution, wherein said buffer is free from calcium and magnesium ions;
e) a fluorescence signal quantifier
The kit allows the quantification of the melanin transferred between cells.
Brief Description of the Accompanying Drawings:
Figure 1: illustrate(s) noise and false signal generated while the estimation of melanin uptake/transfer through quantitative phagocytosis process.
Figure 2: illustrate(s) false signal and noise generated while the estimation of melanin uptake/transfer through fluorescence intensity quantification method.
Figure 3: illustrate(s) a graph showing validation of assay with positive and negative controls of melanin phagocytosis.
Figure 4: illustrate(s) validation of the assay and true quantification by assay of present invention.
Figure 5: illustrate(s) incorrect quantification of melanin transfer assay by microplate reader obtained by use of beads of size 0.5 to 1 micron.
Figure 6: illustrate(s) a graph showing percentage inhibition of melanin transfer.
Figure 7: illustrate(s) graphical representation of the % fluorescence intensity of background noise produced in the routine culture plate. The use of routine tissue culture treated plate led to a high background noise – as much as 56.2% of the total signal.
Figure 8: illustrate(s) photomicrographs of the background noise produced in the routine culture plate of Figure 7. Figure 8a shows the red fluorescent beads scattered and stuck on the routine
culture plate giving high background noise while Figure 8b shows the same microscopic field as in Figure 8a, marked with cell boundaries in green, indicating that the red beads were seen outside the cells stuck on the routine culture plate.
Figure 9: illustrate(s) graphical representation of the % fluorescence intensity of background noise produced by the process of present Invention. The background noise constitutes only 9% of the overall signal produced.
Figure 10: illustrate(s) photomicrographs of the generated background noise produced by the process of present Invention. As evident, the background noise indicated by beads (red dots) are negligible.
Figure 11: illustrate(s) the relation of cell confluency and the background noise.
Figure 12: illustrate(s) the photomicrographs of background noise obtained at confluency level of below 80% as against a predetermined confluency level of >80%. Figure 12a shows maximum red beads scattered outside the cells, stuck on the plate outside the cells, in cell confluency less than 80% while Figure 12b shows less red beads stuck on the plate in cell confluency greater than 80%.
Figure 13: illustrate(s) Brightfield photomicrograph at less than 80% confluency level
Figure 14: illustrate(s) Fluorescent photomicrograph at less that 80% confluency level (showing same microscopic field as figure 13 with background noise beads in interstitial places between cells as red dots).
Figure 15: illustrate(s) % fluorescence intensity obtained from triggered cells (with positive trigger KGF) when cells are incubated and washed with buffer used in the prior art.
Figure 16: illustrate(s) % fluorescence intensity obtained from triggered cells (with positive trigger KGF) when cells are incubated and washed with buffer free from calcium and magnesium ions, of the present Invention.
Figure 17: illustrate(s) fluorescent photomicrograph of background noise obtained in theno-treatment plate. The noise is indicated by red dots scattered all over the image (well-surface)
Detailed Description of the Invention
According to one aspect, the present invention provides an assay and a kit for testing a compound for its role as a promoter or inhibitor of melanin transfer between cell(s). The assay and the kit enables testing the quantity of the melanin uptake/transfer between cells.
In the present embodiment, the invention has been described with reference to the uptake or transfer of melanin between cell(s), however such description should not be considered as restricting the scope of the present invention. Further, it would be possible for a person skilled in the art to practice the present Invention considering the other biomolecule as well without departing from the scope of the present invention.
The assay and the kit of the present Invention has the advantageous property of having high throughput and lesser background noise during the assessment of samples as melanin transfer inhibitor/promoter. The assay and the kit are highly efficient in testing approximately 96 samples/6 to 7 hours without giving any false positive results. The kit of the present invention has also been able to utilize microplate reader for quantifying the fluorescence intensity in determining the quantity of melanin transferred from a cell to other. These technical advantage(s) have been achieved by the specific optimization of the process step(s) and corresponding parts of the kit performing these steps, which include(s) use of carboxylated modified microsphere of size less than 0.5 micron and particularly 0.2 micron, use of the cell(s) at a predetermined cell confluency level in the microtiter plates, use of a medium which is free from calcium and magnesium ions for suspending the modified microsphere, use of wash buffer i.e. phosphate buffered saline free from calcium and magnesium. These aspects are detailed in the following description.
Generally, the in-vitro assay comprises steps of culturing and seeding of the cell(s) i.e. melanocyte or keratinocyte, in a carboxylated modified microtiter plate, which is further incubated for 18 to 24hrs at 370C ± 20C. The incubated cells are further treated with the test compound for determining its role as melanin transfer promoter/inhibitor.

The treated cells are further reacted with carboxylated modified fluorescent microsphere(s)suspended in a medium free from calcium and magnesium ions followed by washing the cells with a buffer solution free from calcium and magnesium ions and thereafter quantifying the fluorescence signal of cell(s) for determining the quantity of transferred melanin between cells.
The detailed steps for the determination of a compound as the melanin transfer promoter/inhibitor between cell(s) are following in which:
a. The cells (keratinocyte and/or a melanocyte) are cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C.
b. The cells resulting from step (a) is treated with the test compound. The confluency level of the cell(s) is preferably greater than 80%.The confluency level of the cell is critical and it significantly affects the background signal / noise ratio. Confluency level of more than 80% with HaCaT keratinocytes is typically achieved by seeding 8000cells per 96 well and allowing its overnight growth. It has been noted that half covered / half confluent well gave the background signal / noise of nearly 85% of the well containing cells.
c. Further, the cells resulting from the above step is incubated with modified fluorescent microspheres (less than 0.5 micron size and particularly 0.2 micron size) suspended in a medium free from calcium and Magnesium ions for about 6 hour.The fluorescent microsphere is of polystyrene beads and is carboxylated modified.
d. Thereafter, washing the cells resulting from above step with a buffer free of calcium and magnesium ions. The use of a buffer which is free from calcium and magnesium ions helps to reduce the background noise.
e. Quantifying the fluorescence signal using microplate reader to determine the tendency of an agent to act as the promoter or inhibitor of the melanin between cells.The complete monolayer in the wells was read using the microplate reader (Excitation: 560 nm; Emission: 590 nm) to quantify the signal from ingested particles. The increase or the strong fluorescence signal indicates the test compound as the promoter of the melanin transfer while the absence or lowfluorescence signalindicates the test compound as the inhibitor of the melanin transfer. The microplate reader helps in quantifying the fluorescence signal which helps in determining the amount of the melanin transferred between cells.

Validation of Assay:
The assay has been further validated using appropriate positive and negative control(s) to test its ability whether it would behave in the same manner as in a melanocyte-keratinocyte system (co-culture). The high throughput process detailed in the present invention was shown to be a valid assay for melanin transfer, in comparison to the established assays which are present in the prior art having low throughput methodology(ies).
To validate the assay, well known triggers and inhibitors of melanin transfer, as reported in the prior art have been used. The trigger agent included PAR-2 agonist peptide, keratinocyte growth factor, alpha-MSH, UV irradiation while inhibiting agent includes Soybean Trypsin Inhibitor, Niacinamide, Genistein, Macelignin etc.
The procedure followed is mentioned below:
a. The test compound is treated with a melanocyte/keratinocyte cell, said cell being cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C. The cell confluency level is critically greater than 80% at this stage. The test compound is an agent which is required here to be tested for its tendency for the transfer of melanin. Also, the other known trigger agent like PAR-2 agonist peptide, keratinocyte growth factor (KGF), alpha-MSH, UV irradiation while inhibiting agent like Soybean Trypsin Inhibitor, Niacinamide, Genistein, Macelignin etc. may be used for verifying the assay.
b. Further, the cells resulting from the above step is incubated with 0.2 micron sized modified fluorescent microspheres suspended in a medium free from calcium and Magnesium ions for about 6 hour.
c. Thereafter, washing the cells resulting from step (c) with a buffer free of calcium and Magnesium ions.
d. Quantifying the fluorescence signal using microplate reader to determine total uptake or transfer of biomolecule.
The result obtained is shown in Figure 3 wherein it was noted that these molecules displayed the same behaviour as in the conventional assay, indicating that the current invention is a valid representation of a method for evaluating the transferred melanin from the cell. KGF and PAR-2 agonist peptide increased the melanin transfer whereas STI and Niacinamide inhibited the melanin transfer compared to control, which validated the assay with respect to the prior art.
Following experiments (the positive and the negative example) were done for testing the effect of the bead(s) size on the background noise and the signal quantification of the process.
Positive Example:
Herein, Beads of size 0.1 – 0.4 microns have been used in the process. Figure 4 shows the correct quantification of melanin inhibitory activity when beads were used in the size of between 0.1 – 0.4 microns. Figure 4 illustrates validation of the assay and true quantification by assay of the present invention. The red fluorescent beads in figure 4 did not stick to the plate and did not cause background noise and were found to be collected in the perinuclear region inside the cells. The microplate reader was able to read the fluorescence signal accurately.
Procedure followed is mentioned below:
a. The test cells are cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C.
b. The cells resulting from step (a) is treated with Par-2 agonist peptide, KGF, STI and niacinamide for uptake at cell confluency levels greater than 80%.
c. Further, the cells resulting from the above step is incubated with 0.2 micron sized modified fluorescent microspheres suspended in a medium free from calcium and Magnesium ions for about 6 hour.
d. Thereafter, washing the cells resulting from step (c) with buffer or saline free of calcium and Magnesium ions
e. Quantifying the fluorescence signal using microplate reader to determine total uptake or transfer of biomolecule.
f. Post-exposure, complete monolayer in the wells was read using the microplate reader (Excitation: 580 nm; Emission: 605 nm) to quantify the signal from ingested bioparticles. The fluorescence reading of the negative control well was checked for negligible background fluorescence to ensure that the signal in other treated wells is representative of ingested bioparticles.

Negative Example:
Experiment was performed considering the size of beads above 0.5 micron (particularly 0.5 – 1 micron).It was seen that the microplate reader did not provide the correct results and also the background noise was also present due to which the estimation was not appropriate. Figure 5 shows pictorial representation of the incorrect quantification of melanin transfer assay by microplate reader obtained by use of beads of size 0.5 – 1 micron. The green color in Figure 5 indicates cell membranes (boundaries) stained with Wheat Germ Agluttinin (WGA) while the red color indicates fluorescent beads. As evident, the red beads posed background noise and were found stuck to the plate (scattered red dots) as well as clustered outside on the cell surface (thick red clusters). Therefore, beads of these sizes led to incorrect quantification of melanin transfer. Figure 5a shows beads of 0.5 micron while figure 5b shows beads of 1 micron.
The procedure followed is mentioned below:
a. The cells are cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C.
b. The cells resulting from step (a) is treated with test compound or the known trigger or inhibit agent wherein the cell’s confluency level should be greater than 80%.
c. Further, the cells resulting from the above step is incubated with 1µ sized modified fluorescent microspheres suspended in a medium free from calcium and Magnesium ions for about 6 hour.
d. Thereafter, washing the cells resulting from step (c) with buffer or saline free of calcium and Magnesium ions
e. Quantifying the fluorescence signal using microplate reader to determine total uptake or transfer of biomolecule.
The Invention in another aspect provides a kit for testing a compound for its role as Trigger/Inhibitor of melanin transfer and for quantifying the melanin transfer/uptake in a cell.
The kit comprises following components:
a) a cell in which the transfer of melanin has to be tested.The cell may be a keratinocyte and/or a melanocyte
b) surface modified microtiter plate. The plate is modified to have the carboxylated groups which helps in obtaining the reduced background signal during the assay
c) modified fluorescent microsphere(s)
d) a Triggering agent/ Inhibiting agent/ test compound for the transfer of melanin;
e) a buffer solution;
f) a fluorescence signal quantifier;
The kit is useful in detecting the transfer of amount of melanin from melanocyte cell and in the estimation of the amount of melanin transferred into keratinocyte(s).

The microtiter plate is modified to contain carboxylate groups. The fluorescent microsphere(s) beads also comprise(s) carboxylate group which is kept suspended in a medium free from calcium and magnesium. The buffer is a phosphate buffer saline free from calcium and magnesium. The phosphate buffered saline has preferable range of pH varying from 7.1 to7.5. Biological buffers with a pH range between 7.1 to 7.5 could be used for this purpose e.g. HEPES buffer, HBSS etc. The fluorescence signal quantifier is preferably a microplate reader. The microplate reader has been used specifically in the present Invention so as to achieve the effective throughput assay.

The signal quantifier is preferably a microplate reader. Other instruments for this purpose may also be used however the results may not be as efficient as to the microplate reader.

The method for evaluating biomolecule uptake / transfer by a cell by the present kit is being detailed below:
a. 8000 HaCaT cells/ per well were seeded in 100ul of medium, in a 96 well microplate and incubated in a CO2 incubator overnight.
b. Post-overnight, when the cells were adhered to the culture dish and attained their morphology, they were treated with active test molecules (herbals/supplier based) in freshly prepared 200ul of culture medium, for 24 h (old 100ul media was completely removed from the wells). The pH of the medium must be checked before use and should be in the range of 7.1-7.5.
c. The fluorescent particles were reconstituted in Hanks’ balanced salt solution (HBSS) so that the stock concentration is 0.1% and sonicated for 15 minutes, in order to disperse the aggregated particles until all the fluorescent particles are homogeneously dispersed.
d. Immediately after sonication the cells were exposed to particles and incubated in CO2 incubator for 6 hours (post-24 h incubation with actives). A negative control well with bioparticles only was also included in the assay.
e. Post-exposure, complete monolayer in the wells was read using the microplate reader (Excitation: 560 nm; Emission: 590 nm) to quantify the signal from ingested bioparticles. Fluorescence reading of the negative control well was checked for negligible background fluorescence to ensure that the signal in other treated wells is representative of ingested bioparticles.

Comparative experimental details:
a) Data for improvement in High throughput achieved by the present Invention:
The assay and kit of the present Invention can screen about 96 samples for the uptake/transfer of melanin in just 6 to 7 hours, as against the existing processes which employs minimum of 28 hours / 12 samples by microscope (counting method) and 16-20 hours / 12 samples by microscope (fluorescent intensity measurement).
Further, the process of the present Invention is highly simple as compared to those of existing method.
Method of the Prior art In-vitro assay of the present Invention
Involves Microscopic imaging which is a cumbersome process The present invention involves only 15 minutes - 1hr post completion of experiment, as it allows the utilization of a fluorescent plate reader to quantify the signal in the absence of any background, without the need for any processing steps.
Post completion of experiment, the processing of samples involves:
1) Fixation of cells in the culture dish with agents such as methanol, formaldehyde etc. - fixation time varies between 1hr to overnight.
2) Imaging of at least 3 visual fields for each sample well, which involves adjustment of magnification, focus, exposure, capture, saving of file - time taken is roughly 3-5 minutes per sample. Therefore for 100 samples, this will be equivalent to 300-500 minutes.
3) Quantification of fluorescence signals on the digital images through manual counting (time depends on extent of bead uptake) or fluorescence intensity measurement (this involves standardizing the software parameters for each experiment, which can be time consuming). No processing steps required in the process of present
Invention

The above table clearly show the simplicity of the process of the present Invention as compared to that of the prior art.
b) Data for noise reduction achieved by the present Invention:
The assay as known in the prior art gives high background noise. The results achieved by the conventional culture plate process for estimating the transfer of melanin in a cell is shown in Figures 7 and 8. The graph in figure 7 and photomicrographs of background noise obtained in Figure 7 shows effect of the use of routine tissue culture treated plate which led to a high background noise as much as about to 56.2% of the total signal. Figure 8 shows the presence of % fluorescence intensity of background noise as 56.2%. Figure 8a shows the red fluorescent beads scattered and stuck on the routine culture plate giving high background noise while Figure 8b shows the same microscopic field as in Figure 8a, marked with cell boundaries in green, indicating that the red beads were seen outside the cells stuck on the routine culture plate.
The process of the present Invention shows % fluorescence intensity of background noise as only 9.4%, which is shown in Figure 9 while figure 10 shows photomicrographs of the generated background noise, which shows the significant reduction (of about 46.8%) in the background noise as compared to the processes existing in the art. As evident from Figure 10, the background noise indicated by beads (red dots) are negligible.
The selection of cell confluency level in the microtiter plate plays a crucial role in the reduction of background noise. The present Invention uses 80-90% cell confluency level which helps in achieving desirable reduction in noise level (Figure 11). Figure 11 shows that more than 80% and preferably 80% to 90% cell confluency level in the well generates the fluorescence intensity of background noise as 52.5% while if cell confluency level is less than 80% then the fluorescence intensity of background noise rises to 84.3%. Figure 11 shows the noise, which is depicted by the signal produced by “background” condition, is much lower in the plate with higher well confluency.
Figure 12 shows the photomicrographs of background noise obtained at confluency level of below 80% as against a predetermined confluency level of >80%. The background noise posed at less than 80% confluency level is shown as fluorescent. The red dots indicate the red fluorescent beads. Figure 12a shows maximum red beads scattered outside the cells, stuck on the plate outside the cells, in cell confluency less than 80% while figure 12b shows less red beads stuck on the plate in cell confluency greater than 80%.
The Brightfield and Fluorescent photomicrographs of the background noise generated at less than 80% confluency level are shown in Figure(s) 13 and 14 respectively.
Figure 13 shows Brightfield photomicrograph at less than 80% confluency level which reveals position of cells on plate while figure 14 shows Fluorescent photomicrograph at less that 80% confluency levels which reveals background noise beads in interstitial places between cells.
It has also been found very surprisingly that incubation of cells with modified beads and washing of cells in a medium free from calcium and magnesium ions drastically reduced the background noise and made the whole process time efficient and cost effective.

Figure 15 shows the signal obtained from triggered cells (with positive trigger KGF) is very much similar to the vehicle control cells, when cells were incubated and washed with buffer as used in the prior art. This time the buffer contained calcium and magnesium. The fluorescence intensity of the vehicle i.e. phosphate buffer saline and of the keratinocyte growth factor was 100% and 109% respectively. Keratin Growth Factor (KGF) showed only a minimum increase of phagocytosis of 109% over vehicle control, contrary to its reported behavior, thus representing false quantification of melanin transfer.
However, when the in vitro assay of the present Invention has been performed, which particularly uses the phosphate buffered saline free of calcium and magnesium, it has been seen that the fluorescence intensity of the triggered cells was 162% as compared to the vehicle i.e. phosphate buffer saline (100%). The result is shown in Figure 16 wherein the KGF showed the expected increase of 162% in melanin transfer representing correct quantification of melanin transfer.

• Culture plate type:
It was observed that when a regular tissue-culture grade culture plate was used, lot of noise and false signal were obtained. In order to circumvent this problem, various culture plates varying in their tissue culture treatment types were tested:
a) No-treatment (neutral) culture plate
b) Ultra-low cell binding culture plate
c) Carboxylated culture plate

Figure 17 shows no-treatment (neutral) culture plate, in which it has been seen that beads were adhered to the culture plate. This adhering of beads causes background noise. The noise is indicated by red dots scattered all over the image (well-surface).
In the Ultra-low cell binding culture plate, neither the cells nor the beads were found to be adhered therefore the signal could not be quantified.
Of the above tested plates, carboxylated culture plate was found to significantly reduce the background noise which is evident from Figures 7 to 10.
The invention will now be explained with the help of following examples. However, the scope of the invention should not be limited to these examples as the person skilled in the art can easily vary the proportion of the ingredients and combinations.

Example 1:
Culturing and seeding of cells into surface modified micro titer plate:
The cells are cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C. HaCaT keratinocyte cell line was procured from NCCS, Pune and maintained in Dulbecco’s Modified Eagle’s Medium-Nutrient mixture Ham’s 12 (DMEM-F12) medium supplemented with 1% Penicillin & Streptomycin and 10% FBS in a CO2 incubator with 5% CO2. The cells were routinely checked and kept free of bacteria, fungus and mycoplasma contamination. Thereafter 8000 HaCaT cells/ per well were seeded in 100ul of medium, in a 96 well microplate and incubated in a CO2 incubator overnight.
Example 2:
Incubation of cells with the test compound/Trigger/Inhibitor of the melanin transfer:
The cells resulting from the above example is treated with the test compound. Instead of the test compound, well known trigger/Inhibitor may also be used for checking the quantity of the melanin uptake/ transferred in a cell or from a cell. The cell confluency level is greater than 80%. Further, post-overnight, when the cells were adhered to the culture dish and attained their morphology, they were treated with active molecules (herbals/supplier based) in freshly prepared 200ul of culture medium, for 24 h.This level of confluency with HaCaT keratinocytes is typically achieved by seeding 8000cells per 96 well and allowing for overnight growth. It has been noted that half covered / half confluent well gave the background signal / noise of nearly 85% of the well containing cells.

Example 3:
Treating the cells with fluorescent microsphere:
The cells resulting from the above example were exposed to the fluorescent microspheres (less than 0.5 micron size) suspended in a medium free from calcium and Magnesium ions for about 4 to 24 hours followed by Incubation in CO2 incubator for 4 to 24 hours (preferably post-24 h incubation with actives i.e. test compound). A negative control well with the active i.e. the test compound only was also included in the assay.

Example 4:
Washing of cells:
The cells were washed with a buffer free from calcium and magnesium ions.It was noted that suspending the microspheres in and washing the incubated cells with a buffer free from calcium and magnesium ions was critical in determining the sensitivity and reproducibility of the assay. When cells were washed with a buffer or saline without Caand Mg ions, higher signal to noise ratio was obtained as compared to washing with a buffer or saline containing Ca and Mg ions. The results are shown in figure 15 and 16.

Example 5:
Quantifying the fluorescence signal using microplate reader to determine total uptake or transfer of biomolecule:
Post-exposure, complete monolayer in the wells was read using the microplate reader (Excitation: 560 nm; Emission: 590 nm) to quantify the signal from ingested bioparticles. The fluorescence reading of the negative control well was checked for negligible background fluorescence to ensure that the signal in other treated wells is representative of ingested bioparticles.

Example 6:
The assay has been used for testing the melanin-transfer inhibitory activity of certain plant species. The plant species taken for the test include(s) Asparagus racemosus, Aphanimixisrohituka, Kappaphycus and Acanthophoraspicifera.
The procedure followed is mentioned below:
a. The test cells are cultured and seeded into surface modified 96 or more welled microtiter plates followed by incubation at about 37º C.
b. The cells resulting from step (a) is treated with Asparagus racemosus, Aphanimixisrohituka, Kappaphycus and Acanthophoraspicifera for uptake at cell confluency levels greater than 80%.
c. Further, the cells resulting from the above step is incubated with 0.2 µ sized modified fluorescent microspheres suspended in a medium free from calcium and Magnesium ions for about 6 hour.
d. Thereafter, washing the cells resulting from step (c) with buffer or saline free of calcium and Magnesium ions
e. Quantifying the fluorescence signal using microplate reader to determine total uptake or transfer of biomolecule.

The test revealed thatAsparagus racemosus, Aphanimixisrohituka, KappaphycusandAcanthophoraspicifera exhibit significant melanin transfer inhibition at 50 ppm while no significant melanin transfer inhibitory activity at or below 5 ppm has been found.
Asparagus racemosus, Aphanimixisrohituka, KappaphycusandAcanthophoraspicifera when used at 50 ppm provide 9, 7, 20 and 16% melanin transfer inhibition respectively as shown in table 1 and Figure 6.
Table 1
Plant species % Inhibition of melanin transfer
Vehicle Control 0
A. racemosus 50 ppm 9
A. rohituka 50 ppm 7
Kappaphycus10 ppm 20
A. spicifera 10 ppm 16
A. racemosus 5 ppm 0
A. rohituka 5 ppm 2
Kappaphycus 1 ppm 2
A. spicifera 1 ppm 1

Example 7:
A method for evaluating melanin uptake/ transfer in a cell comprises following steps:
1) Culturing and seeding keratinocyte cells (HaCaT /primary keratinocyte) into carboxylate modified 96-well plates and incubating overnight at 37º C.
2) Treating the cells with triggers / inhibitors of phagocytosis at >80% confluency levels.
3) Incubating the cells with carboxylate-modified fluorescent microspheres (polystyrene beads) of 0.2 micron, suspended in a medium free from calcium and Magnesium ions for 6h.
4) Multiple washing with phosphate buffered saline free of calcium and Magnesium ions.
5) Quantifying the fluorescence signal by microplate reader Quantifying the fluorescence signal using microplate reader to determine total uptake /transfer on the same day. The signal is to be quantified without dislodging the monolayer or harvesting the cells by trypsinization / lysis, using the bottom reading parameter of the instrument.