F 7000 spectrofluorometer

The UV-2910 Hitachi spectrophotometer (Hitachi, Tokyo, Japan) was applied to collect the UV absorption spectra. The OVA samples (0.5 mg/ml) were scanned from 240 to 320 nm at a scan speed of 800 nm/min. The F-7000 Hitachi spectrofluorometer (Hitachi, Tokyo, Japan) was used to measure the intrinsic fluorescence of OVA (0.2 mg/ml). The emission spectra were scanned from 300 to 420 nm at a speed of 1,200 nm/min with the excitation wavelength of 280 nm. The bandwidth, excitation, and emission slits were all 5.0 nm.

The fluorimetric and electrochemiluminescent methods were employed to evaluate the biodegradation efficiency of naphthalene, phenanthrene, and pyrene. The fluorescence determination method is based on highly efficient quantum fluorescence of hydrocarbons possessing conjugated aromatic rings (Schwarz and Wasik 1975 (link)). In order to determine the excitation and emission wavelengths, the excitation and fluorescence spectra for naphthalene, phenanthrene, and pyrene were obtained by using commercial PAH standards in hexane at a concentration of 10⁻⁴ mol/l. The wavelength of excitation radiation (λ_exc) for naphthalene, phenanthrene, and pyrene was at 275, 348, and 340 nm, accordingly. Afterward, the PAH standard solutions in hexane were used to carry out measurements of fluorescence intensity at analytical wavelengths of λ_em = 323, 384, and 386 nm, and the calibration curves were obtained.
Since hydrocarbons in the solid state also exhibit intense fluorescence (the undissolved molecules in the exciting beam of the fluorimeter may considerably interfere with the measurement), a threefold extraction of samples with the use of 100 ml of hexane was carried out (5 min). The measurements were carried out using a Hitachi F-7000 spectrofluorometer.

Parasites were purified and loaded with Fura 2-AM as described^{64 (link),65 (link)}. An Hitachi F-7000 spectrofluorometer was used for calcium measurements. Conditions and calibration were as published^{65 (link),66 (link)}.
For ER calcium measurements, cells were resuspend to a final density of 1 × 10⁹ cells/ml in HBS buffer (135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl₂, 11.6 mM HEPES, 1.5 mM CaCl₂, 11.5 mM glucose) containing 1 mg/ml BSA, 0.2 mg/ml of pluronic F127 and 20 μM Mag-Fluo-4-AM. Loading was for 60 min in the dark at room temperature. Subsequently, parasites were washed 3 times and the cell pellet resuspended in 1.8 ml CLM (20 mM NaCl, 140 mM KCl, 20 mM PIPES, pH 7.0) containing 1 mM EGTA at 1 × 10⁹ cells/ml. Cells were permeabilized with 30 μg/ml digitonin for 5 min. Permeabilized cells were washed 3 times with CLM containing 1 mM EGTA and resuspended to a final density of 1 × 10⁹ cells/ml and kept in ice. For each test, 50 μl (5 × 10⁷) of the suspension was added into 1.95 ml of CLM containing 1 mM EGTA with 375 µM CaCl₂ (220 nM free Ca²⁺).

Fluorescence experiments were performed on the Hitachi F 7000 spectrofluorometer (Tokyo, Japan), with a programmable temperature controller attached. The protein solution was excited at 295 nm and the emission was recorded in the wavelength range of 300 nm to 500 nm. The PMT voltage was set at 500 V and the excitation and emission slits were set at 5 nm.