2.2.2. BNZ activity against intracellular amastigote forms
BNZ activity against amastigote forms was performed by seeding 1 ×
104 Vero cells·well–1 in 24-well
plates with rounded coverslips at 37º C in humidified 95 % air, 5 %
CO2 atmosphere in Gibco RPMI 1640 medium supplemented
with 10% (v /v ) hiFBS. After 24h of incubation, Vero cells
were infected with culture-derived trypomastigotes at a multiplicity of
infection (MOI) ratio of 10:1. After 24 h of infection, nonphagocyted
trypomastigotes were washed, and infected Vero cells were treated by
adding BNZ at a concentration range from 100 to 0.4 μM (500
μL·well–1 volumes) at 37º C in humidified 95 % air,
5 % CO2 atmosphere for 72 h. Untreated growth controls
were also included. Counting of amastigotes and infected cells was
performed in methanol-fixed and Giemsa-stained preparations by random
analysis of 500 host cells (Martín-Escolano et al. , 2020b). The
infectivity index, defined as the average number of amastigote forms in
infected cells multiplied by the percentage of infected cells, was also
determined. The trypanocidal activity was expressed as the
IC50 and IC90 (inhibitory concentrations
50 and 90) using GraphPad Prism 6 software. Each BNZ concentration was
tested in triplicate in four separate determinations.