To set the new assay up, the drugs currently marketed for the chemotherapy of Chagas disease were selected as reference compounds: BZ and NFX. Pure drugs were provided by Roche, Brazil, Bayer and Germany, respectively, and they were dissolved in dimethyl sulfoxide (DMSO; Stanton®, Buenos Aires, Argentina) in stock solutions at 20 mg/ml. DMSO concentration never exceeded 0.2% in plate wells, with no effect on the host cell viability nor parasite replicative growth [16 (link)]. Roswell Park Memorial Institute (RPMI)-1640 with or without phenol red (Sigma; R6504 or R8755, respectively). Sodium bicarbonate (Emeve®; P1551-500). Gentamicin (Sigma; G1397). Penicillin–streptomycin (Sigma; P4333). Resazurin solution (Sigma, R7017). CPRG (Roche Diagnostics GmbH; 10 884 308 001). Nonidet™ P-40 substitute (Roche Diagnostics GmbH; 11 754 599 001). Heat-inactivated, UV-irradiated foetal bovine serum (FBS) (Natocor® or Internegocios S.A, Argentina). Geneticin (G418; Life Technologies).
Protocol detail
Chagas Disease Chemotherapy Protocol
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Assay
Cell viability
Chagas disease
Chemotherapy
Cprg
Diagnostics
Dmso
Drugs
Foetal bovine serum
G418
Geneticin
Gentamicin
Nonidet p 40
Parasite
Penicillin
Replicative
Resazurin
Sodium bicarbonate
Streptomycin
Corresponding Organization : University of Buenos Aires
Other organizations : Instituto de Biología Molecular y Celular de Rosario, National University of Rosario, Western University
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Variable analysis
independent variables
- Concentration of the drugs BZ and NFX
- Parasite replicative growth
- Host cell viability
- DMSO concentration (never exceeded 0.2% in plate wells)
- RPMI-1640 with or without phenol red
- Sodium bicarbonate
- Gentamicin
- Penicillin–streptomycin
- Heat-inactivated, UV-irradiated foetal bovine serum (FBS)
- Geneticin (G418)
- Positive control: Not specified
- Negative control: Not specified
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