Ls55 spectrofluorimeter

The intrinsic fluorescence resulting from the presence of aromatic amino acids (tryptophan and tyrosine) of Apo-B100 was used to study apoprotein properties using emission spectra (excitation wavelength = 295 nm). The position of the maximum emission of tryptophan fluorescence is sensitive to the hydrophobicity of the surrounding environment. The samples of untreated LDLs and modified LDLs were resuspended in 5 mM Hepes buffer, 150 mM NaCl at pH 7.4. The emissions spectra were evaluated using a Perkin Elmer LS 55 spectrofluorimeter. All measurements were conducted in triplicate [51 (link)].

The assay was performed on a LS55 spectrofluorimeter (Perkin-Elmer, Leatherhead, UK) using 96-well white polystyrene microtiter plates (Porvair Sciences, Leatherhead, UK) according to a method described by Fredotovic et al. [73 (link)] and Nazlić et al. [74 (link)]. Each reaction contained 180 µL of fluorescein (1 µM), 70 µL 2,2′-Azobis(2-methyl-propionamidine) dihydrochloride (AAPH, Acros Organics, Fair Lawn, NJ, USA) (300 mM), and 30 µL of plant extract or reference standard Trolox (6.25–50 µM) (Sigma–Aldrich, St. Louis, MO, USA). All experimental solutions were prepared in a phosphate buffer (0.075 mM, pH 7.0). The extract of essential oil was prepared in acetone (20 mg/mL). This solution was further diluted 400× with the phosphate buffer for the experiments. For the hydrosol analyses we used total hydrosol diluted 10x. Measurements were performed in triplicate. ORAC values for essential oil were expressed as µmol of Trolox equivalents (TE) per g of essential oil and for hydrosol as µmol of Trolox equivalents (TE) per L of hydrosol.

The measurement of the low-temperature fluorescence spectrum (77 K) was performed according to the methodology of Wiciarz et al. [56 (link)]. The frozen plant material was homogenized to leaf powder and then mixed with Hepes 0.05 M + 0.33 M sorbitol buffer (pH 7.5) in the proportion of 0.1 g/1 mL. The measurement was performed at 77 K using an LS 55 spectrofluorimeter (Perkin Elmer, Waltham, MA, USA). The sample was placed in a quartz glass capillary and excited with radiation at a wavelength of 437 nm. The fluorescence spectrum was recorded in the range of 650–800 nm using the FL WinLab 4.0 software (Perkin Elmer, Waltham, MA, USA). The curves were normalized to the maximum PSII fluorescence value. Based on the spectra, the values of the F730/F690 ratio were calculated, which allows to estimate the relative share of PSI and PSII in the thylakoid membranes.

CRC assays were performed on freshly isolated mitochondria from adult non-tg mouse brain and heart (n = 6) after GNX-4728 was administered intravenously by tail vein injection (15 mg/kg in 20% DMSO and 40% PEG400) followed by a survival of 5 min. Control mice (n = 6) were injected with vehicle. Brain and heart mitochondria were isolated using a similar procedure as described (Wong et al., 2013 (link)). Mitochondrial CRC was assessed fluorimetrically in the presence of the fluorescent Ca²⁺ indicator Calcium Green 5N (Invitrogen Molecular Probes) using a temperature controlled Perkin-Elmer LS 55 spectrofluorimeter as described (Fancelli et al., 2014 (link)). Briefly, purified organ mitochondria were pulse-loaded with 10 mM calcium and then challenged with increasing concentrations of calcium until mitochondrial permeability transition was triggered as evidenced by complete release of mitochondrially-stored calcium due to mPTP opening.

Synaptosomal pellets were analyzed for glutamate release using enzyme-linked fluorescence method previously described [48 (link)]. In brief, synaptosomes were suspended in hepes-buffered medium containing 2 mM NADP⁺, 50 units of glutamate dehydrogenase, and 1.2 mM CaCl₂, and the synaptosome suspension was stimulated after 5 min with either 1 mM 4-AP or 15 mM KCl. Increases in fluorescence due to production of NADPH was determined (λ excitation = 340 nm and λ emission = 460 nm) with a PerkinElmer LS55 spectrofluorimeter. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg). Values quoted in the text and depicted in bar graphs represent the levels of glutamate cumulatively released after 5 min of depolarization, and are expressed as nmol/mg/5 min.

Measurement of [Ca²⁺]_C was determined by fluorescent probe fura-2 AM. Synaptosomes were incubated with 5 µM fura-2in the hepes-buffered medium containing 0.1 mM CaCl₂ for 30 min at 37 °C. After fura-2 loading, the synaptosomes were centrifuged for 1 min at 3000× g and pellets were resuspended in the hepes-buffered medium containing 1.2 mM CaCl₂. Fura-2/Ca fluorescence was monitored in a PerkinElmer LS55 spectrofluorimeter (PerkinElmer Life Sciences, Waltham, MA, USA) at 340 nm and 510 nm. [Ca²⁺]_C (nM) was calculated using the equations described previously [50 (link)].

Synaptosomal pellets were analyzed for glutamate release using enzyme-linked fluorescence method previously described [24 (link),26 (link)]. As synaotosome is not amenable to electrical stimulation, a number of biochemical depolarization protocols have been developed, including the use of K⁺ channel blocker 4-AP or high external [K⁺] [24 (link)]. Therefore, synaptosomes were suspended in hepes-buffered medium containing 2 mM NADP⁺, 50 units of glutamate dehydrogenase, and 1.2 mM CaCl₂, and the synaptosome suspension was stimulated with either 1 mM 4-AP or 15 mM KCl. Increases in fluorescence due to production of NADPH was determined using a PerkinElmer LS55 spectrofluorimeter with excitation and emission wavelengths set at 340 nm and 460 nm, respectively. Released glutamate was calibrated by a standard of exogenous glutamate (5 nmol) and expressed as nanomoles glutamate per milligram synaptosomal protein (nmol/mg protein). Values quoted in the text and depicted in bar graphs represent the levels of glutamate cumulatively released after 5 min of depolarization, and are expressed as nmol/mg protein/5 min.