A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein.
Convenient and economical assays capable of screening many compounds are vital to advance the development of drug therapy. This is particularly important for many of the infections that occur mainly in the Third World. The development of such a spectrofluorometric assay for the protozoan parasite Leishmania is presented here. Using multimeric (four monomers) green fluorescent protein (GFP), Leishmania amazonensis promastigotes were generated with brightness measurable in 96-well microtiter plates. The promastigotes maintained the parental characteristics, were infective to murine macrophages and to mice, and the level of GFP fluorescence corresponded to the number of inoculated cells. The feasibility of using this assay for testing drugs kinetically and in a concentration-dependent manner, under microplate culture condition, was demonstrated with amphotericin B and the herbicide oryzalin, respectively. This assay is the first to allow a real-time analysis of antileishmanial agents with live promastigotes. The method of expressing multimeric GFP for in vitro drug screening is likely to be extendable to many species of parasitic protozoa.
Chan
Marion Man-Ying
MM
Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Room 509 OMS, Philadelphia, PA 19140, USA. marionc@astro.temple.edu
Bulinski
J Chloe
JC
Chang
Kwang-Poo
KP
Fong
Dunne
D
D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D004136:Dinitrobenzenes / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007891:Leishmania / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D008018:Life Cycle Stages; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D021261:Parasitic Sensitivity Tests / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013424:Sulfanilamides; D014162:Transfection
D004136:Dinitrobenzenes; D008164:Luminescent Proteins; D013424:Sulfanilamides; D049452:Green Fluorescent Proteins; C012465:oryzalin; D000666:Amphotericin B