title: Fluorescent ester dye-based assays for the in vitro measurement of Neospora caninum proliferation.
abstract: Techniques for the measurement of parasite loads in different experimental models have evolved throughout the years. The quantification of stained slides using regular cytological stains is currently the most common technique. However, this modality of evaluation is labor-intensive, and the interpretation of the results is subjective because the successes of the assays mainly rely on the abilities of the professionals involved. Moreover, the novel genetic manipulation techniques that are commonly applied for closely related Toxoplasma gondii have not yet been developed for Neospora caninum. Thus, we aimed to develop a simple protocol for parasite quantification using pre-stained N. caninum tachyzoites and fluorescent probes based on ester compounds (i.e., CFSE and DDAO). For this purpose, we employed a quantification procedure based on flow cytometry analysis. Pre-stained parasites were also examined with a fluorescent microscope, which revealed that both dyes were detectable. Direct comparison of the numbers of CFSE+ and DDAO+ cells to the values obtained with classical cytology techniques yielded statistically comparable results that also accorded with genomic DNA amplification results. Although the fluorescence emitted by DDAO was more intense and provided better discrimination between the populations of parasitized cells, CFSE+ tachyzoites were detected for several days. In conclusion, this study describes a simple, fast, low-cost and reproducible protocol for N. caninum quantification that is based on parasite pre-staining with fluorescent ester-based probes.
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Caroline M
authors.initials: CM
authors.lastname: Mota
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Marcela D
authors.initials: MD
authors.lastname: Ferreira
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Lourenço F
authors.initials: LF
authors.lastname: Costa
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Patrício S C
authors.initials: PS
authors.lastname: Barros
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Murilo V
authors.initials: MV
authors.lastname: Silva
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: Fernanda M
authors.initials: FM
authors.lastname: Santiago
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil.
authors.forename: José R
authors.initials: JR
authors.lastname: Mineo
authors.affiliation: Laboratório de Imunoparasitologia "Dr. Mário Endsfeldz Camargo", Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia, Av. Amazonas s/n, Bloco 4C, Sala 4C01, Campus Umuarama, 38405-320 Uberlândia, Minas Gerais, Brazil. Electronic address: tiagomineo@icbim.ufu.br.
authors.forename: Tiago W P
authors.initials: TW
authors.lastname: Mineo
mesh_terms: D049109:Cell Proliferation; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D018688:Neospora / Q000166:cytology* / Q000502:physiology; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes
keywords: CFSE; DDAO; HeLa; Macrophages; Neospora caninum; Parasite load