Dp2 camera

HeLa cells (30,000 per slide) were grown on coverslips, then washed in phosphate buffered saline (PBS), fixed with 2% formaldehyde (in PBS) for 15 min at RT and blocked with 10% fetal calf serum in PBS for 1 h. The cells were then incubated with 25 μg PvCelTOS in the absence or presence of 40833, 40835, or 40838 synthetic peptide (>99% purity) in a 1:100 (protein-peptide) molar ratio for the competition assay at 4°C overnight. The coverslips were washed twice with PBS and then incubated with anti-histidine monoclonal Ab (mAb) (Sigma) at 1:4,000 dilution for 1 h at RT. Following two washes with PBS, the cells were incubated with monoclonal FITC-conjugated anti-mouse secondary Ab. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and fluorescence was visualized by fluorescence microscope (Olympus BX51) using an Olympus DP2 camera and Fiji software. HeLa cells without protein or with histidine peptide (HHHHHH) were incubated with primary and secondary Abs as control.

COS-7 (American Type Culture Collection CRL-1651) cells were cultured in Dulbecco-Modified Eagle Medium (DMEM, Gibco, Co Dublin, Ireland) supplemented with 10% foetal calf serum (Gibco) at 37 °C with 5% CO₂; 2 × 10⁴ COS-7 cells were then seeded and incubated in chamber slide sterile wells until reaching 40–60% confluence. Transfection involved using 300 ng PvMSP10-C, PvDBP-RIII or pHVDR22 plasmids with FuGENE HD reagent (Promega), following the manufacturer’s protocol. The cells were cultured in OptiMEM medium for 48 h in a humidified atmosphere containing 5% CO₂ in an incubator at 37 °C. Immunofluorescence assays evaluated transfection efficiency. Briefly, transfected COS-7 cells were fixed with PBS containing 2% formaldehyde for 15 min at RT, blocked with 10% foetal calf serum (in PBS) for 1 h at RT and incubated with anti-DL6 Ab (Santa Cruz, Dallas, TX, USA) in 1:1000 dilution overnight at 4 °C and then with fluorescein-conjugated goat anti-mouse Ab for 1 h 30 min at RT in the dark. Each step involved three washes with PBS. Cell nuclei were stained with 0.1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 20 min; fluorescence was visualised on an Olympus BX51 microscope using an Olympus DP2 camera and Fiji software. Transfection efficiency percentage was calculated as total amount of fluorescent COS-7 cells × 100/total amount of COS-7 cells counted in 30 fields.