Guava easycyte mini flow cytometer system

Log-phase promastigotes parasites were incubated in HPMI buffer (5×10⁶/mL) (20 mM HEPES, 132 mM NaCl, 3.5 mM KCl, 0.5 mM MgCl₂, 5 mM glucose, 1 mM CaCl₂, pH 7.4) containing 0.3% (w/v) BSA and 10 µM of NBD-PC or NBD-PE for 30 minutes at 25°C [21] (link). Before the addition of NBD-phospholipids, parasites were incubated for 15 minutes in buffer containing 500 µM PMSF to inhibit the catabolism of phospholipids. After labelling with NBD, parasites were washed twice with ice-cold HPMI containing 0.3% BSA to remove short-chain NBD-phospholipids from the external plasma membrane [22] (link), [23] (link) and then finally resuspended in PBS for flow cytometry analysis.
Labeled parasites were analysed at room temperature using Guava EasyCyte Mini Flow Cytometer System (Millipore). Data from 5,000 cells, defined by gating at data acquisition, was collected and analysed using CytoSoft version 4.2.1 software (Guava Technologies) and FlowJo version 9.4.9 software (Tree Star, Ashland, Oregon).
Alternatively, 1×10⁶ labeled parasites (5×10⁶/mL in PBS) at room temperature were analysed in a microplate reader (POLARstar Omega, BMG Labtech) with excitation at 460 nm and emission at 530 nm. At least three independent experiments were done in duplicate.

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).