Infection of Macrophages with L. donovani Parasites

For infection experiment, equal number of THP1 and hMDM (~10⁶) were used for two experimental setups. In one set, the adhered THP1 cells and hMDM were infected with either wild-type (WT), ISP2KD, or ISP2OE Leishmania parasites at parasite/macrophage multiplicities of 10:1. Cultures were kept at 37°C in 5% CO₂ for 12 h. The unbound parasites were removed by washing with RPMI without FBS. The cells were harvested (12 h after infection) from each group, washed with PBS and kept at −80°C for RNA and protein isolation. In other set, macrophages (THP1 and hMDM) were pretreated with wortmannin (200 nM) for 3 h and then infected with WT parasites. Untreated macrophages were infected with WT, ISP2KD, and ISP2OE parasites. Infected cells were kept in incubator and cells were harvested (after 12 h) for protein isolation and the supernatant was collected for ELISA and kept at −80°C. The parasite load was measured both in THP1 cells and hMDM at 24 h after infection by counting the number of intracellular amastigotes per 100 macrophages and the rate of infection was also analyzed in both cells (THP1 and hMDM) by counting the percent infected macrophages after Giemsa staining.