LiRecDT1-GFP binding was evaluated as described above, except that B16-F10 cells (0.5 × 103 cells) were incubated
with 10 μg/mL of the recombinant fluorescent toxin (5 h, 37 °C). Non-specific binding of GFP alone to the cells was evaluated as a negative control. For binding competition assays, the fluorescence protocol was the same as described above, except that B16-F10 cells were previously incubated with an excess of LiRecDT1 (100 μg/mL) for 1 h at 37 °C Cyclopamine purchase and then with 10 μg/mL LiRecDT1-GFP. The samples were observed using a Zeiss Axio Observer.Z1 inverted microscope (Carl Zeiss, Germany). Single images were obtained using a 63× oil lens for differential interface contrast (DIC) microscopy and a monochromatic camera (AxioCam HRm, Carl Zeiss) to examine fluorescence intensity. Finally, AxioVision LE software was used for image processing and morphometric measurements in the Zeiss image format ABT 888 (ZVI). B16-F10 cells (1 × 108 cells/mL) were prepared in Ringer’s Solution (122.5 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 10 mM HEPES, 11 mM glucose, 1 mM NaH2PO4, pH 7.4) containing 5 mM CaCl2 and treated according to Kaestner et al. (2006) and Haase et al. (2009). B16-F10 cells were loaded with Fluo-4 AM (10 μM) in buffer with Pluronic F-127 (0.01%) for 30 min at 37 °C. This indicator exhibits high-affinity binding to Ca2+ (Kd = 345 nM)
and shows a large increase in fluorescence intensity in response to Ca2+ binding (>100 fold). Subsequently, the cells were washed not twice with Ringer’s Solution and equilibrated for de-esterification for 30 min at room temperature. Then, the cells were incubated with 25 μg/mL recombinant phospholipase-D (LiRecDT1) for 5, 15, 30, 45, 60 or 90 min.
Cells incubated under the same laboratory conditions but in the absence of phospholipase-D for 90 min were used as a control. Following this reaction, the cells were transferred to Black 96-well plates at a density of 1 × 106 cells/well in a total volume of 200 μL, and the resulting fluorescence was recorded on a Tecan Infinite M200 spectrofluorometer (Tecan) using an excitation wavelength of 485 nm and measuring emission at 535 nm. Additionally, Fluo-4 dye-loaded B16-F10 cells were allowed to settle onto coverslips, and images of calcium-dependent fluorescence were obtained using an Axio Observer.Z1 inverted microscope Zeiss (Carl Zeiss, Germany). Fluo-4 AM was excited at 488 nm, with emission detected using an LP 505 nm filter (green channel). Single images were obtained using a 63× oil lens for differential interface contrast (DIC) microscopy and a monochromatic camera (AxioCam HRm, Zeiss, Carl Zeiss, Germany) to measure the fluorescence intensity. Finally, AxioVision LE software was used for image processing and to perform morphometric measurements in the Zeiss image format (ZVI).