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Antileishmanial Bioassays

96-Well Micro Titer Plate Bioassay.
Leishmania Promastigotes are grown in bulk early in modified NNN biphasic medium using normal physiological saline. Leishmania parasite Promastigotes were cultured with RPMI 1640 medium supplemented with 10% heat inactivated foetal bovine serum (FBS).   Parasites at log phase are centrifuged at 2000 rpm for 10 minutes, and washed three times with saline at same speed and time.  Parasites are diluted with fresh culture medium to a final density of 106 cells/ml.
In a 96-well micro titer plate, 180ml of medium was added in different wells. 20ml of the experimental compound was added in medium and serially diluted. 100ml of parasite culture was added in all wells.  Two rows should be left for negative and positive control.  Negative controls receives medium while the positive control contains varying concentrations of standard antileishmanial compound e.g. Amphotericin B, Pantamidine. The plate was incubated between 21-22 °C for 72 hrs. The culture was examined microscopically on an improved neubauer counting chamber and IC50   values of compounds possessing antileishmanial activity were calculated by Software Ezfit 5.03 Perella Scientific.  All assays were run in duplicate.