Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo

Zhi Xiu Lin, J. R S Hoult, Amala Soumyanath

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56 Citations (Scopus)

Abstract

A rapid 96-well plate assay using sulphorhodamine B (SRB) protein stain for cell number has been adopted to screen herbs used in traditional treatments of vitiligo for substances capable of stimulating melanocyte proliferation. Its applicability to melan-a cells, a mouse pigmented cell line, has been validated. SRB assay produced good linearity up to 11 x 104 cells/well and interference by melanin present in the cells accounted for less than 10% of the total optical density readings. The intra-assay variation was small but interassay variation was marked. For better assay precision, it is recommended that the results to be compared should be performed on the same day and controls should be plated in the same experiment, ideally in the same plate. Optimum conditions for exponential melan-a cell growth were established: viz. initial plating density (3-8 x 103 cells/well), incubation period (4 days) and foetal bovine serum concentration (5%). Under these conditions cells were responsive to the mitogen tetradecanoyl phorbol acetate (TPA). Out of 28 herbal extracts screened in this assay, significant stimulation (P <0.05) of melanocyte proliferation was observed, in the absence of TPA, using aqueous extracts of Astragalus membranaceous root, Citrus reticulata peel, Dictamnus dasycarpus root bark, Ophiopogon japonicus root, Poria cocos sclerotium and Tribulus terrestris fruit.

Original languageEnglish (US)
Pages (from-to)141-150
Number of pages10
JournalJournal of Ethnopharmacology
Volume66
Issue number2
DOIs
StatePublished - Aug 1999
Externally publishedYes

Fingerprint

lissamine rhodamine B
Vitiligo
Drug Evaluation
Melanocytes
Cell Line
Dictamnus
Ophiopogon
Acetates
Tribulus
Citrus
Melanins
Mitogens
Reading
Fruit
Coloring Agents
Cell Count

Keywords

  • Herbal screening
  • Melan-a cell line
  • Melanocyte
  • Proliferation
  • SRB assay
  • Vitiligo

ASJC Scopus subject areas

  • Pharmacology

Cite this

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title = "Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo",
abstract = "A rapid 96-well plate assay using sulphorhodamine B (SRB) protein stain for cell number has been adopted to screen herbs used in traditional treatments of vitiligo for substances capable of stimulating melanocyte proliferation. Its applicability to melan-a cells, a mouse pigmented cell line, has been validated. SRB assay produced good linearity up to 11 x 104 cells/well and interference by melanin present in the cells accounted for less than 10{\%} of the total optical density readings. The intra-assay variation was small but interassay variation was marked. For better assay precision, it is recommended that the results to be compared should be performed on the same day and controls should be plated in the same experiment, ideally in the same plate. Optimum conditions for exponential melan-a cell growth were established: viz. initial plating density (3-8 x 103 cells/well), incubation period (4 days) and foetal bovine serum concentration (5{\%}). Under these conditions cells were responsive to the mitogen tetradecanoyl phorbol acetate (TPA). Out of 28 herbal extracts screened in this assay, significant stimulation (P <0.05) of melanocyte proliferation was observed, in the absence of TPA, using aqueous extracts of Astragalus membranaceous root, Citrus reticulata peel, Dictamnus dasycarpus root bark, Ophiopogon japonicus root, Poria cocos sclerotium and Tribulus terrestris fruit.",
keywords = "Herbal screening, Melan-a cell line, Melanocyte, Proliferation, SRB assay, Vitiligo",
author = "Lin, {Zhi Xiu} and Hoult, {J. R S} and Amala Soumyanath",
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doi = "10.1016/S0378-8741(98)00199-8",
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T1 - Sulphorhodamine B assay for measuring proliferation of a pigmented melanocyte cell line and its application to the evaluation of crude drugs used in the treatment of vitiligo

AU - Lin, Zhi Xiu

AU - Hoult, J. R S

AU - Soumyanath, Amala

PY - 1999/8

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N2 - A rapid 96-well plate assay using sulphorhodamine B (SRB) protein stain for cell number has been adopted to screen herbs used in traditional treatments of vitiligo for substances capable of stimulating melanocyte proliferation. Its applicability to melan-a cells, a mouse pigmented cell line, has been validated. SRB assay produced good linearity up to 11 x 104 cells/well and interference by melanin present in the cells accounted for less than 10% of the total optical density readings. The intra-assay variation was small but interassay variation was marked. For better assay precision, it is recommended that the results to be compared should be performed on the same day and controls should be plated in the same experiment, ideally in the same plate. Optimum conditions for exponential melan-a cell growth were established: viz. initial plating density (3-8 x 103 cells/well), incubation period (4 days) and foetal bovine serum concentration (5%). Under these conditions cells were responsive to the mitogen tetradecanoyl phorbol acetate (TPA). Out of 28 herbal extracts screened in this assay, significant stimulation (P <0.05) of melanocyte proliferation was observed, in the absence of TPA, using aqueous extracts of Astragalus membranaceous root, Citrus reticulata peel, Dictamnus dasycarpus root bark, Ophiopogon japonicus root, Poria cocos sclerotium and Tribulus terrestris fruit.

AB - A rapid 96-well plate assay using sulphorhodamine B (SRB) protein stain for cell number has been adopted to screen herbs used in traditional treatments of vitiligo for substances capable of stimulating melanocyte proliferation. Its applicability to melan-a cells, a mouse pigmented cell line, has been validated. SRB assay produced good linearity up to 11 x 104 cells/well and interference by melanin present in the cells accounted for less than 10% of the total optical density readings. The intra-assay variation was small but interassay variation was marked. For better assay precision, it is recommended that the results to be compared should be performed on the same day and controls should be plated in the same experiment, ideally in the same plate. Optimum conditions for exponential melan-a cell growth were established: viz. initial plating density (3-8 x 103 cells/well), incubation period (4 days) and foetal bovine serum concentration (5%). Under these conditions cells were responsive to the mitogen tetradecanoyl phorbol acetate (TPA). Out of 28 herbal extracts screened in this assay, significant stimulation (P <0.05) of melanocyte proliferation was observed, in the absence of TPA, using aqueous extracts of Astragalus membranaceous root, Citrus reticulata peel, Dictamnus dasycarpus root bark, Ophiopogon japonicus root, Poria cocos sclerotium and Tribulus terrestris fruit.

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