Dec 06, 2024
In vitro human tyrosinase inhibitory assay (human melanoma cell lysate based)
- Gengxuan Shi1
- 1Institute of Biomedicine and Glycomics, Griffith University
Protocol Citation: Gengxuan Shi 2024. In vitro human tyrosinase inhibitory assay (human melanoma cell lysate based). protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxw97dv8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 04, 2024
Last Modified: December 06, 2024
Protocol Integer ID: 113708
Abstract
Tyrosinase (TYR) inhibitors are required to treat skin hyperpigmentation. Currently live cell-based TYR assays and mushroom TYR in vitro assays are the common methods used to screen for TYR inhibitors. However, these methods are either time consuming and expensive or are not human TYR (hsTYR) specific. Here, we describe a simple hsTYR assay using cell lysate prepared from pigmented human melanoma cell lines that takes less than 3 hours to complete after collecting cell pellets. We confirmed the assay is species specific by using a known hsTYR inhibitor, kojic acid, as a positive control, while arbutin, which inhibits mushroom TYR, but not hsTYR, was not effective. This assay is a simple method to confirm hsTYR inhibition before conducting follow-up studies in live biological models.
Materials
Cell lines and materials
MM418C1 is a clone of the MM418 melanoma cell line that originated from the primary lesion of the cutaneous surface (Clark et al., 1994 & Fechner et al., 1994).
Reagents
L-Glutamine, penicillin/streptomycin, trypsin, RPMI 1640 medium (powder) were purchased from Life Technologies (CA, USA). Bovine serum albumin (BSA), 3,4-Dihydroxy-L-phenylalanine (L-Dopa), sodium phosphate, kojic acid, Triton X-100 and T75 flask were purchased from Sigma Chemicals (NSW, Australia). Fetal bovine serum (FBS) was purchased from Bovogen Biologicals (VIC, Australia). Trypan blue 0.4% solution was purchased from MP biomedicals (VIC, Australia). The DC protein assay kit was purchased from Bio-Rad (NSW, Australia)
Safety warnings
Proper PPE must be worn all the time.
Risk assessment for handling chemicals and equipment use must be undertaken.
In vitro human tyrosinase inhibitory assay (human melanoma cell lysate based)
In vitro human tyrosinase inhibitory assay (human melanoma cell lysate based)
2h 25m
2h 25m
Seed human melanoma MM418C1 cells (or equivalent melanoma cell line) in a T75 cell culture flask and grow with RPMI 1640 medium, which contains 10% (v/v) fetal bovine serum (FBS), 200 mM L-glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin (Sigma chemicals, Australia) until 80% confluent.
Mix 10 µL of cell suspension with 10 µL of 0.4% (w/v) trypan blue solution, then apply 10 µL of the mixture onto a clean hemocytometer and count the cells.
10m
Collect 3 x 106 cells (can be stored at -80 °C for up to at least 672:00:00 without loss of tyrosinase activity) by trypsinization and centrifugation at 300 x g, 25°C, 00:05:00 .
10m
Lyse the cell pellet using 600 µL Triton X-100 lysis buffer (1% (v/v) Triton X-100, 50 mM sodium phosphate buffer, 6.8 , Sigma chemicals, Australia) and sonicate for 00:00:30 On ice using Epishear Sonicator (CA, USA).
5m
Collect the supernatants by centrifugation at 7500 x g, 4°C, 00:30:00 .
30m
Determine the protein concentration of the clarified cell lysate with the DC protein assay kit (Bio-Rad, Australia)
15m
For each sample, 20 µL of the potential tyrosinase inhibitors (1 mg/mL) were added to a 96-well plate in triplicate, followed by 160 µL of 15 mM L-3,4-dihydroxyphenylalanine(L-Dopa) solution in 0.1 M sodium phosphate buffer (0.049 M Na2HPO4•7H2O, 0.051 M NaH2PO4•H2O). Finally, 20 µL of the clarified cell lysate was added to each well.
10m
20 µL of 1 mg/mL kojic acid + 160 µL L-DOPA + 20 µL cell lysate is used as the positive control,
20 µL of 1% (v/v) Triton X-100 lysis buffer + 160 µL L-DOPA + 20 µL cell lysate is used as the blank.
Measure the change in absorbance at λ475 nm over 01:00:00 using a CLARIOstar multi-mode plate reader (BMG Labtech, Germany) and normalize the data by using the protein concentration obtained in step 6.
1h
Tyrosinase activity (% remaining) can be calculated using the following equation:
Tyrosinase activity (%) = [(A1-A2) / (A3-A4)] * 100%
L-DOPA solution and sodium phosphate buffer were used as a blank (A1)
L-DOPA and cell lysate without test sample were used as a negative control (A2)
L-DOPA and cell lysate with the test sample were the test reactions (A3)
L-DOPA and the test sample without cell lysate were used as a negative control (A4)
5m
Protocol references
Clark, J., Grabs, A. J., Parsons, P. G., Smithers, B. M., Addison, R. S., & Roberts, M. S. (1994). Melphalan uptake, hyperthermic synergism and drug resistance in a human cell culture model for the isolated limb perfusion of melanoma. Melanoma Research, 4(6), 365-370.
Fechner, G. A., Michel, J., Sturm, R. A., Jacobs, J. J., & Parsons, P. G. (1994). Reduction of DNA synthesis, pigment synthesis, pigmentation gene mRNA and resistance to UVB in human melanoma cells treated with analogues of a histamine (H2) agonist. Biochemical pharmacology, 48(1), 121-130.
Acknowledgements
Griffith University Postgraduate Research Scholarship (GUPRS)
Griffith University International Postgraduate Research Scholarship (GUIPRS)
Professor Peter Parsons from QIMR Berghofer Research Institute (Brisbane, Australia) for providing the human melanoma cell line