Figure 3

Identification of well-expressing Leishmania strains. A) Four IFP-His and three ANAC42-His fusion protein-expressing Leishmania cell lines (clones 4, 5, 11, 12 and 1, 2, 3, respectively) of different volumes (2 μL, 10 μL and 100 μL) were transferred into the wells of a 96-well ELISA plate and centrifuged for 2 min at 2,500 g, followed by pipeting again 100 μL of the respective cell lines into neighbouring wells indicated by a white-dashed box. IR scanning was performed using the Odyssey Infrared Imaging System. B) Schematic outline of the previously established LEXSY protocol and the IFP protocol described here. The flow-chart high-lights the differences of the two Leishmania protein expression protocols in terms of expenditures of time and effort. C) Twelve different IFP fusion protein (1, ARR1-IFP-His; 2, IFP-ARR1-His; 3, ANAC42-IFP-His; 4, IFP-ANAC42-His; 5, TPK1-IFP-His; 6, IFP-TPK1-His) expressing Leishmania cell lines were picked from the selective plate and transferred into the wells of a 96-well microtiter plate filled with 150 μL BHI medium, followed by two days of incubation and IR scan (upper panel). The complete volumes of all cell lines were transferred into the wells of a 24-well deep-well plate containing 1 mL BHI medium, followed by incubation for two days. 100 μL-samples of each cell line were IR-scanned in the wells of a 96-well microtiter plate (lower panel). IFP-His expressing cell line was used as positive control (P) and ANAC42-His expressing cell line as negative control (N).