Victor x3 fluorometer

L. amazonensis promastigote forms were prepared and standardize as described previously (item 2.8.1). In addition, 1 μL of Wortmannin (Sigma–Aldrich^®, 1 mg/mL), a known autophagy inhibitor, was added in the treated samples (Blommaart et al., 1997 (link)). After to add 10 μL of monodansylcadaverine (MDC; Sigma–Aldrich^®) at dark, the samples were incubated for 1 h, at 37°C, for posteriorly analysis on Perkin-Elmer Victor X3 fluorometer, at λ_ex = 380 nm e λ_em = 525 nm (Pincus et al., 1975 (link)).

Parasites were prepared and standardize in accordance to item 2.8.1. Next, 5 μL of Nile Red (Sigma–Aldrich^®) 1 mg/mL methanol were added to treated parasites and incubated during 30 min, at dark and room temperature. Fluorescence was measured on Perkin-Elmer Victor X3 fluorometer, at λ_ex = 485 nm e λ_em = 535 (Greenspan et al., 1985 (link)). Nile Red is a phenoxazone dye that fluoresces intensely at yellow–gold color in the presence of triacylglycerols, cholesterol, or cholesterol esters, considered neutral lipids (Fowler and Greenspan, 1985 (link)).

Intrasynaptosomal glutamate and glutamine content was measured with Glutamine and Glutamate Determination Kit according to manufacturer’s instructions (Sigma-Aldrich, Germany).
Glutamate release from synaptosomal preparations was measured by fluorometric assay essentially as described by Nicholls et al. (1987 (link)). Briefly, ~100 μl synaptosomes (~1 mg/ml) suspended in aCSF supplemented with 2 mM NADP⁺ and 6.32 U L-glutamic acid dehydrogenase (and 2 mM CaCl₂ whenever appropriate) was distributed into each of the 96 wells. Synaptosomes were depolarized with 30 mM KCl 5 min thereafter and the increase in NADPH fluorescence was monitored over a 10 min time period. Fluorescence emission was recorded at 450 nm and the excitation wavelength was set at 360 nm. Each experimental condition was carried out in triplicate on the same plate using Victor X3 fluorometer (Perkin Elmer, USA). The amount of released glutamate was calculated based on calibration curves prepared in parallel and the enzyme lag (Nicholls et al., 1987 (link)) was accounted for when converting rates to nmol/mg protein/10 min. The results obtained in the presence of 2 mM CaCl₂ reflect total glutamate release whereas in the absence of Ca²⁺ in the reaction mixture as Ca²⁺-independent glutamate release.