Nucleofector 2b machine

The 100 µl of blood was collected from B. microti-infected mice (10–20% parasitemia) into a 1.5 ml Eppendorf tube containing heparin, washed twice with incomplete RPMI media. Human T-cells nucleofector kit (VAPA-1002, Lonza, Basel, Switzerland) was used for the parasite transfection. First, 18 µl of solution ‘A’ of the kit and 82 µl of solution ‘B’ of the kit were mixed in a tube and kept on the ice. The 10 µg of the linearized construct was added to the solution. Next, 20 µl of packed RBC with 10% parasitemia were added to the transfection mixture per transfection. The transfection was performed using the manufacturer’s U033 program in a Nucleofector-2B machine (Lonza, Basel, Switzerland) and 100 µl of incomplete RPMI (Gibco, Thermo Fisher Scientific, India) was added to the transfected parasites. The transfected parasites were immediately injected (Intravenous) into the mice. In a control experiment a plasmid without homologous arms and without a reporter gene was used as a control. After 48 h, the transfected parasites were analyzed by fluorescence microscopy to visualize GFP expressing parasites. The GFP expressing parasites were sorted by FACS and immediately injected into a mouse to enrich the transfected parasites.

Transfection was performed through electroporation as previously described [11 (link)]. Briefly, 1×10⁶ cells were transfected with 4 ug plasmids (pDsRed-C1, pDsRed-C1-LA or pDsRed-C1-PG) through Amaxa NHDF Nucleofector kit (F-09376; Lonza) on a Nucleofector 2b machine (Lonza). Cells were then either seeded in 6 well plates for western blot analysis or in chamber slides for immunofluorescent staining. Doxorubicin treatment and subsequent assays were performed at 72h after transfection.