Ca 074 me

Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS) and penicillin-streptomycin (10,000 U and 10,000 mg/ml) were obtained from Invitrogen (Bleiswijk, Netherlands). The WST-1 reagent was purchased from Roche Applied Science (Vilvoorde, Belgium). NaCl 0,9% was obtained from B. Braun Medical (Diegem, Belgium), Triton X-100 from Flucka (Buchs, Switzerland), the cathepsin B inhibitor CA-074-Me from Bachem (Switzerland). Methanol, dimethyl sulfoxide (DMSO), Tris buffered saline, Tween 20, lipopolysaccharide (LPS), cytochalasin D, ATP and chloroform were purchased from Sigma-Aldrich, 2-mercaptoethanol, Laemmli Sample Buffer from Bio-Rad (Hercules, USA) and hydrofluoric acid (HF) from Merck (Darmstadt, Germany).

1×10⁶ BMDC/ml were harvested at day 7 of culture, plated in 6-well plates and incubated overnight at 37°C, 5% CO₂. For some experiments, BALB/c BMDC were pre-incubated with 10 µM CA074Me (Bachem, Bubendorf, Switzerland), 10 µM CLIK148 (kindly provided by Prof. Tanja Schirmeister), or 10 µM of Z-Arg-Leu-Arg-α-aza-glycyl-Ile-Val-OMe (ZRLR, kindly provided by Dr. Timo Burster, University of Ulm, and Dr. Ewa Wieczerzak, University of Gdansk) for 4 hours prior to infection. eGFP-L. major promastigotes were harvested, washed 3 times in warm PBS, added to the BMDC at a 1∶5 infection ratio, and further incubated at 37°C. After 2 hours of exposure of the BMDC to the parasites, the cells were washed with warm PBS and resuspended in fresh medium at a concentration of 1×10⁶ cells/ml. Part of the cells was fixed in paraformaldehyde (PFA, 4%; Applichem, Darmstadt, Germany). The remaining cells were incubated for a total of 4 hours or 24 hours post infection, fixed, and the amount of infected cells at the different time points was determined by flow cytometry, together with the expression of maturation markers as described next.