Selection procedure for induction of PMM-resistance using intracellular <i>L. donovani</i> amastigotes.

<p>Late stationary-phase promastigotes were used to infect primary mouse macrophages exposed to 2-fold PMM dilutions starting from 500 µM. After 5 days, surviving intracellular amastigotes at the highest PMM concentration (checked after Giemsa staining on a duplicate plate) were allowed to transform back into promastigotes by replacing the RPMI cell culture medium by MEM-based promastigote medium and incubation at room temperature for 1 week. Next, the recovered promastigotes were expanded in 25 ml tissue culture bottles without PMM pressure and used to infect a new batch of primary mouse macrophages for another cycle of selection.