Ls 50b spectrofluorometer

The hydrophobicity of LPS aggregates was determined by using the pyrene fluorescence peak I to peak III ratio method. Briefly, an apyrogenic phosphate-buffered saline saturated with the pyrene probe (Sigma-Aldrich) was filtered and added to LPS solution in saline (final LPS concentration, 20 μmol/L), and fluorescence was measured at room temperature on an LS50B spectrofluorometer (Perkin Elmer, Waltham, MA, USA). Pyrene emission fluorescence spectra were scanned from 350 to 400 nm with an excitation wavelength of 335 nm. The hydrophobic ratio was calculated by dividing the intensity of the first fluorescence peak (peak I–374 nm) by that of the third peak (peak III–384 nm). Hydrophobicity correlates inversely with the I–III ratio.

Cytosolic Ca²⁺ levels were determined spectrofluorometrically using the probe FURA-2 AM (Del Pino et al., 2019a (link),b (link)). Olive pollen (100 mg) from sub-samples stored in the dark at 5°C was suspended in 10 ml PBS and hydrated for 2 days at 25°C. Hydrated pollens were harvested by centrifugation at 1,000 g × 4 min and then resuspended in 2 ml HBSS buffer (120 mM NaCl, 5.0 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, 25 mM Hepes, pH 7.4). Pollen suspensions were incubated in the dark with FURA-2 (2 μl of a 2-mM solution in DMSO) for 120 min, followed by centrifugation at 1,000 g × 4 min. Pollens were then harvested and suspended in ~10 ml of HBSS containing 0.1 mM EGTA, which was included to rule out or, at least, minimize a potential background due to contaminating ions (to obtain a suspension of 1 × 10⁶ of pollen granules hydrated per ml). Fluorescence was measured in a Perkin-Elmer LS 50 B spectrofluorometer (ex. 340 and 380 nm, em. 510 nm), set with a 10-nm and a 7.5-nm slit width in the excitation and emission windows, respectively. Fluorometric readings were taken after 300–350 s. When required, samples of pollen, H₂O₂ and Meg were added for specific purposes, as described in the Results section. Cytosolic calcium concentrations [(Ca²⁺)_c] were calculated as described in Grynkiewicz et al. (1985) (link).

The effects of the pCBs on intracellular Ca²⁺ release that was induced by TRPV1 activation was determined using the selective intracellular fluorescent probe Fluo-3 AM (Molecular probes, Eugene, OR, USA). The HaCaT cells (1 × 10⁶ cells) were loaded for 15 min at 25 °C with 4 µM of Fluo-3 AM, which contained 0.02% Pluronic F-127 (Molecular probes) in an OPTIMEM medium, and then they were washed in the Tyrode’s buffer (145 mM of NaCl, 2.5 mM of KCl, 1.5 mM of CaCl₂, 1.2 mM of MgCl₂, 10 mM of D-glucose and 10 mM of HEPES; pH 7.4), resuspended in 2 mL of the Tyrode’s buffer and transferred into the quartz cuvette of the LS50B spectrofluorometer (Perkin Elmer, Waltham, MA, USA). Fluorescence was measured at 25 °C (excitation at λ = 488 nm; emission at λ = 516 nm) from the HaCaT cells, which had been pre-incubated with each pCB (6.0 µM of CBG, 4.0 of µM CBC, 9.0 µM of THCV and 13.0 µM of CBGA) and then stimulated with the selective TRPV1 agonist capsaicin (1 µM), as reported in [64 (link)]. The TRPV1-mediated intracellular Ca²⁺ elevation was expressed as fluorescence intensity (arbitrary units, AU) per 10⁶ cells.

Unilamellar DPPC liposomes were obtained by sonification of 2 mM/L of phospholipid suspension in the same buffer solution as used in DSC experiments (pH 7.4) using a UP 200s sonificator (Dr. Hilscher, GmbH, Berlin, Germany).
Fluorescent dyes: laurdan and prodan stock solutions (1 mM) were prepared in DMSO. The stock solutions of the studied compounds (30 mM) were also prepared in DMSO. The dispersion of DPPC liposomes was incubated with the fluorescent dye in darkness for 30 min at room temperature, then the studied compound was added, and liposomes were incubated for another 20 min (also in darkness at room temperature). In all of the experiments, the final DPPC concentration was 200 μM. The concentration of the fluorescent dye (laurdan or prodan) was 5 μM. The studied compound concentration in the samples was 25–125 μM. The fluorescence experiments were performed with an LS 50B spectrofluorometer (Perkin-Elmer Ltd., Beaconsfield, UK) equipped with a xenon lamp using emission and excitation slits of 5 nm. The excitation wavelength for laurdan was 390 nm and for prodan was 360 nm. The recorded fluorescence spectra were processed with FLDM Perkin-Elmer 2000 software. It was investigated before the measurements that the studied compounds alone did not exhibit fluorescence in the spectral region of interest.

Intracellular ROS accumulation in HMC-1 cells was measured by staining cells with the green fluorescence probe H₂DCFDA, which is rapidly oxidized to highly fluorescent DCF in the presence of intracellular H₂O₂, and analyzed spectrofluorometrically (model Axiovert 200). Briefly, HMC-1 cells (1 × 10⁵ cells/sample) were preloaded with 5 μM H₂DCFDA for 30 min and washed twice with culture medium. The washed cells were incubated with or without SPs or PAF for up to 30 min at 37 °C in a CO₂ incubator. The production of intracellular ROS was determined on a Perkin Elmer LS50B spectrofluorometer using excitation and emission wavelengths of 485 and 530 nm, respectively. All background fluorescence was subtracted using the appropriate controls.

Cells were washed with PBS and then lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 10 mM EGTA, 1 mM EDTA, 10 mM DTT, 1% (v/v) Triton X-100, for 30 min at 4 °C. After centrifugation at 15,000× g for 15 min at 4 °C, supernatants were collected and used for detection of caspase activity.
Cell lysate (60 µg of proteins) was mixed with 20 μM fluorogenic caspase-3 peptide substrate, Ac-DEVD-AMC, in the reaction buffer (50 mM Tris–HCl, pH 7.4, 10 mM EGTA, 1 mM EDTA, 10 mM DTT). The reaction mixture was incubated for 30 min, at 37 °C.
Fluorescence was measured on a Perkin-Elmer LS-50B spectrofluorometer, with excitation at 380 nm and emission at 460 nm.