Cytosoft 4

L. amazonensis promastigotes (5×10⁶/mL) were treated with 30 and 60 µM raloxifene for 20 min or 2 h in M199 medium, supplemented with 10% FCS at 25°C. Parasites treated with 25 µM digitonin were used as a positive control. Untreated parasites and parasites incubated with the highest volume of diluent (DMSO 0.6%) were used as negative controls. Parasites were stained with 10 µM propidium iodide (PI) and immediately analysed by flow cytometry using a Guava EasyCyte Mini Flow Cytometer System (Millipore). A total of 5,000 events were acquired in the region previously established as corresponding to the parasites. Fluorescence was quantified using the CytoSoft 4.2.1 software (Guava Technologies Inc., Hayward, CA, USA). Histograms were drawn using FlowJo software, version 9 for Macintosh (Tree Star, Inc., Ashland, OR).

After removal of the apical and basal media, Caco–2 cells were rinsed with PBS and dissociated with trypsin/EDTA solution (Invitrogen) supplemented with 1.9 mmol/L sodium EDTA (Sigma-Aldrich) for 8 min to get a single-cell suspension. Trypsin was inhibited by adding culture media and the cells were washed with PBS. After resuspension in PBS the cells were analyzed by flow cytometry (Guava easyCyte mini, Millipore, Hayward CA, USA) and the software CytoSoft 4.2.1 (Guavatechnologies, Hayward CA, USA). The arithmetic mean of fluorescence intensity determined for 5,000 cells was used to calculate the mean fluorescence intensity (MFI) of three independent experiments. The MFI of cells incubated without PF–488 labeled peptides was used to subtract background staining.

Triplicate or sextuplet cultures of both cell lines were subjected to time-course experiments. Every 24 h, cultures were washed in PBS, trypsinized, pelleted by centrifugation at 500g for 5 min, and resuspended in PBS. Each sample volume was measured and 25 μl of each sample was combined with 225 μl of ViaCount reagent (Guava Technologies, Hayward, CA), resulting in a 1:10 dilution. The samples were then counted using the Guava ViaCount application in the Guava EasyCyte Mini microcapillary cytometer (Guava Technologies). The Guava ViaCount assay provides an absolute number of cell count by drawing cells into a capillary flow cell of known dimensions at a precisely controlled rate for specific amounts of time. The absolute cell counts depend on the dilution of the suspension, as well as of the total volume of sample from which the aliquot was taken. The data is both, acquired and analyzed, using the CytoSoft 4.1 software (Guava Technologies).