title: Flow cytometric screening for anti-leishmanials in a human macrophage cell line.
abstract: High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC(50)'s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445±0.0005μg/ml, 0.1203±0.018mg/ml, and 26.71μM using THP-1 cells, and 0.179±0.035μg/ml, 0.1948±0.0364mg/ml, and 13.77±10.74μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.
authors.affiliation: Division of Infectious Diseases, University of California, San Diego, CA 92103, USA. srmehta@ucsd.edu
authors.forename: Sanjay R
authors.initials: SR
authors.lastname: Mehta
authors.forename: Xing-Quan
authors.initials: XQ
authors.lastname: Zhang
authors.forename: Roberto
authors.initials: R
authors.lastname: Badaro
authors.forename: Celsa
authors.initials: C
authors.lastname: Spina
authors.forename: John
authors.initials: J
authors.lastname: Day
authors.forename: Kwang-Poo
authors.initials: KP
authors.lastname: Chang
authors.forename: Robert T
authors.initials: RT
authors.lastname: Schooley
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology* / Q000633:toxicity; D000967:Antimony Sodium Gluconate / Q000494:pharmacology* / Q000633:toxicity; D000981:Antiprotozoal Agents / Q000494:pharmacology* / Q000633:toxicity; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005562:Formazans; D006801:Humans; D007894:Leishmania mexicana / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects / Q000469:parasitology*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000633:toxicity; D013778:Tetrazolium Salts; D014343:Trypan Blue
chemical_list: D000981:Antiprotozoal Agents; D004396:Coloring Agents; D005562:Formazans; D013778:Tetrazolium Salts; D010767:Phosphorylcholine; C079782:MTT formazan; C039128:miltefosine; D000666:Amphotericin B; D014343:Trypan Blue; D000967:Antimony Sodium Gluconate