Expression of nanoluciferase in transgenic TcCOL-NLuc parasites.

(A) Luciferase activity was measured in 5 x10³ live TcCOL-NLuc and TcCOL-wt cells using the Nano-Glo^® Live Cell Assay System. (B) Luciferase activity was measured in lysates of 10⁵ TcCOL-NLuc and TcCOL-wt cells using the Nano-Glo^® Luciferase Assay System. Left panel: NLucexpression evaluated in TcCOL-NLuc epimastigotes after passage 1 (P1) and passage 22 (P22) in LIT media containing G418. Right panel: NLuc expression evaluated in tissue culture-derived TcCOL-NLuc trypomastigotes after passage 1 (P1) and passage 5 (P5) in LLC-MK2 cells. (C) Luciferase activity in amastigotes was evaluated in LLC-MK2 cells infected with 5 x10⁴ TcCOL-NLuc or TcCOL-wt trypomastigotes. Four days post-infection, luminescence was assessed in cell lysates using the Nano-Glo^® Luciferase Assay System. (D) Nanoluciferase protein expression in intracellular amastigotes was evaluated in methanol-fixed LLC-MK2 cells infected with 5 x10⁴ TcCOL-NLuc (top panels) or TcCOL-wt (bottom panels) trypomastigotes by immunostaining with rabbit anti-NLuc antibodies (Anti-NLuc). DAPI indicates DNA staining. Values in (A), (B) and (C) represent means ± s.d. of three replicate wells. Statistical differences between groups were calculated using an unpaired t-test with a 95% confidence interval. p-values are indicated. Scale bar: 50 μm.