Arasite suspension in different concentrations, in triplicate. After 48 h of incubation

Arasite suspension in different concentrations, in triplicate. After 48 h of incubation, the parasites were counted and compared to the controls containing parasites in the absence of drugs. The drug concentration corresponding to 50 of the parasite growth inhibition was expressed as IC50. Three independent experiments were performed to confirm the results. Data are presented as mean and 95 CI.Intracellular amastigote assayL. (L.) amazonensis and L. (V.) braziliensis promastigotes with 7? days and 5? days of growth, respectively, were harvested from cultures and added to a fresh Schneider’s medium supplemented with 5 FCS, pH 6.0, and incubated at 32uC for 7 days for L. (L.) amazonensis and 4? days for L. (V.) braziliensis until a complete transformation to amastigote-like. Peritoneal macrophages from BALB/c mice were harvested by washing with ice-cold RPMI 1640 medium, 4 days after induction with a 3 thioglycollate solution. Macrophages were diluted in RPMI 1640 medium (Sigma, Poole, United Kingdom) plus 10 FCS and plated in 24 well plates with a circular cover glass at a plating density of 26105 macrophages/well. Macrophages were allowed to adhere for 2 h at 37uC and 5 CO2, when the medium was replaced by a fresh one and incubated overnight. Macrophages were infected with amastigote-like parasites from L. (L.) amazonensis or L. (V.) braziliensis. The parasites were counted in a Neubauer’s chamber and adjusted to a macrophageamastigote ratio of 1:8. Infected cultures were maintained at 37uC and 5 CO2. After 4 h, extracellular parasites were removed by washing, and a fresh medium containing either TPM compounds, no drug, or the reference drug. AmB (FungizoneH-Bristol- Meyers Squibb Pharmaceutics Ltda, Bedford, USA) was the chosen reference drug and was used at 0.2 mg/ mL. After 72 h, the cover glass was removed from the well, washed in RPMI 1640 medium, set in NT 157 manufacturer microscope blades, fixed with methanol, and stained with Giemsa for evaluation. Under the immersion microscope, infection indexes (number of amastigotes/ 1006 percentage of infected macrophages) were determined by counting the numbers of intracellular amastigotes in 100 macrophages. The experiments were considered valid only when the control group (without drugs) displayed at least 80 of infection. Each point was tested in triplicate and three independent experiments were then performed. Results are presented as mean and 95 CI [25].Materials and Methods Ethics statementThis study has been approved by Ethics Committee for Animal Experimentation from University Federal of Minas Gerais (CETEA/UFMG: 12/2009).The University Federal of Minas Gerais adheres to the standards as outlined by relevant national (CONCEA – Brazilian Government Council for Control of Animal Experimentation) and international guidelines for care and use of laboratory animals.ParasitesPromastigotes of L. (L.) amazonensis (IFLA/BR/1967/PH-8), L. (V.) braziliensis (MHOM/BR/75/M2903), and L. (L.) major (MHOM/IL/80/Friedlin) were maintained at 23uC in Schneider’s Drosophila medium (Merck, Germany) supplemented with 20 heat-inactivated fetal calf serum (FCS) (Gibco, 307538-42-7 Eggenstein, Germany), pH 7.2. The same strain of L. (L.) amazonensis was used for both in vitro and in vivo experiments.Triphenylmethane compounds (TPM)Novel TPM were synthesized by reacting aromatic substrates with 4,49bis (diethylaminobenzophenone) in the 12926553 presence of phosphorus oxychloride in a calorimeter bomb at 140uC under pressure. TPM formul.Arasite suspension in different concentrations, in triplicate. After 48 h of incubation, the parasites were counted and compared to the controls containing parasites in the absence of drugs. The drug concentration corresponding to 50 of the parasite growth inhibition was expressed as IC50. Three independent experiments were performed to confirm the results. Data are presented as mean and 95 CI.Intracellular amastigote assayL. (L.) amazonensis and L. (V.) braziliensis promastigotes with 7? days and 5? days of growth, respectively, were harvested from cultures and added to a fresh Schneider’s medium supplemented with 5 FCS, pH 6.0, and incubated at 32uC for 7 days for L. (L.) amazonensis and 4? days for L. (V.) braziliensis until a complete transformation to amastigote-like. Peritoneal macrophages from BALB/c mice were harvested by washing with ice-cold RPMI 1640 medium, 4 days after induction with a 3 thioglycollate solution. Macrophages were diluted in RPMI 1640 medium (Sigma, Poole, United Kingdom) plus 10 FCS and plated in 24 well plates with a circular cover glass at a plating density of 26105 macrophages/well. Macrophages were allowed to adhere for 2 h at 37uC and 5 CO2, when the medium was replaced by a fresh one and incubated overnight. Macrophages were infected with amastigote-like parasites from L. (L.) amazonensis or L. (V.) braziliensis. The parasites were counted in a Neubauer’s chamber and adjusted to a macrophageamastigote ratio of 1:8. Infected cultures were maintained at 37uC and 5 CO2. After 4 h, extracellular parasites were removed by washing, and a fresh medium containing either TPM compounds, no drug, or the reference drug. AmB (FungizoneH-Bristol- Meyers Squibb Pharmaceutics Ltda, Bedford, USA) was the chosen reference drug and was used at 0.2 mg/ mL. After 72 h, the cover glass was removed from the well, washed in RPMI 1640 medium, set in microscope blades, fixed with methanol, and stained with Giemsa for evaluation. Under the immersion microscope, infection indexes (number of amastigotes/ 1006 percentage of infected macrophages) were determined by counting the numbers of intracellular amastigotes in 100 macrophages. The experiments were considered valid only when the control group (without drugs) displayed at least 80 of infection. Each point was tested in triplicate and three independent experiments were then performed. Results are presented as mean and 95 CI [25].Materials and Methods Ethics statementThis study has been approved by Ethics Committee for Animal Experimentation from University Federal of Minas Gerais (CETEA/UFMG: 12/2009).The University Federal of Minas Gerais adheres to the standards as outlined by relevant national (CONCEA – Brazilian Government Council for Control of Animal Experimentation) and international guidelines for care and use of laboratory animals.ParasitesPromastigotes of L. (L.) amazonensis (IFLA/BR/1967/PH-8), L. (V.) braziliensis (MHOM/BR/75/M2903), and L. (L.) major (MHOM/IL/80/Friedlin) were maintained at 23uC in Schneider’s Drosophila medium (Merck, Germany) supplemented with 20 heat-inactivated fetal calf serum (FCS) (Gibco, Eggenstein, Germany), pH 7.2. The same strain of L. (L.) amazonensis was used for both in vitro and in vivo experiments.Triphenylmethane compounds (TPM)Novel TPM were synthesized by reacting aromatic substrates with 4,49bis (diethylaminobenzophenone) in the 12926553 presence of phosphorus oxychloride in a calorimeter bomb at 140uC under pressure. TPM formul.