Fp 8300 fluorescence spectrometer

The phase composition, crystal structure and crystallinity were examined by a Rigaku Miniflex XRD instrument using Cu Kα radiation (λ = 1.54056 Å). The UV–vis diffused reflectance spectra were analysed by a UV–vis spectrophotometer (JASCO 750) using BaSO₄ as the reflectance reference. The photoluminescence (PL) emission spectra were investigated by a JASCO-FP-8300 fluorescence spectrometer with an excitation wavelength of 330 nm. The chemical composition and vibrational modes of the ZFO samples were analysed by JASCO FTIR-4600. A ZEISS SUPRA 55 was used for FESEM analysis. Morphology and microstructure of the prepared catalysts were investigated by a TEM-JEOL-2010 200 kV instrument. The electrochemical analysis was carried out using an Ivium potentiostat. Photoelectrochemical (PEC) measurements were performed in a Pyrex electrochemical set up, which includes ZFO samples (deposited on FTO) as working photo anode, Ag/AgCl as reference electrode and Pt as counter electrode. The electrolyte chosen for the study was 0.1 M Na₂SO₄ aqueous solution of pH 6.8. A 400 nm cut-off filter was used for the light irradiation during linear sweep voltammetry (LSV) analysis.

MCF-7 cells were cultured in a 6-well plate at initial densities of 500,000 cells/well (2 mL/well) for 24 h (37℃, 5% CO₂). Subsequently, porphyrin compound solutions (1.0 mM in DMSO) were added (20 μL/well) and incubated for 2, 6, 16, and 24 h.
After incubation, the culture medium was removed, and MCF-7 were washed with PBS three times. SDS buffer (10 wt%) was added to the cells for lysis and the cell lysate was centrifuged (10,000 rpm, 15 min). The fluorescence intensity of the supernatant of the cell lysate was recorded using a JASCO FP-8300 fluorescence spectrometer.
The excitation wavelength was 420 nm. The accumulation concentration of porphyrin in MCF-7 cells was measured by using a calibration curve from the fluorescence intensity.

Lipid films containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (43.4 mg, 50 μmol) were prepared by the evaporation of chloroform solutions in glass tubes. The suspension was hydrated with 2 mL of PBS and vortexed for 5 min. The mixture was centrifuged for 5 min at 15,000 rpm, 25 °C, and the supernatant (containing smaller vesicles) was discarded. An equivalent volume of buffer solution was added, and the pellet was re-suspended. This procedure was repeated at least twice to prepare multi-lamellar vesicles (MLV).
To liposome suspension (2 mL) was added to the porphyrin solution (20 μL, 1.0 mM in DMSO) and incubated for 1 h. Then the MLV suspension was centrifuged for 15 min at 15,000 rpm, and the supernatant was removed and 10 wt% SDS buffer (1 mL,) was added to the sediment to destroy the membrane. The fluorescence intensity of porphyrin in the solution was measured using a JASCO FP-8300 fluorescence spectrometer.

Fluorescence spectra were obtained with an FP-8300 fluorescence spectrometer (JASCO, IJsselstein, The Netherlands). Tertiary structure and thermal stability of filgrastim were evaluated based on the intrinsic fluorescence, by using a temperature ramp from 25°C to 70°C with 5°C increments. Filgrastim samples were kept at 300 μg/ml, except Biocilin (250 μg/ml). A 0.3 cm pathlength SUPRASIL grade quartz cell (Hellma Analytics, Müllheim, Germany) was used to record the emission spectra between 290 and 450 nm with a 0.5 nm sampling interval, an excitation wavelength of 280 nm, emission and excitation bandwidths of 5 nm and a scan speed of 500 nm/min. The baseline spectrum was subtracted from each sample spectrum. As not all excipients of filgrastim products are specified by the manufacturers, Neupogen placebo formulation (50 mg/ml sorbitol, 0.04 mg/ml PS 80, 0.59 mg/ml sodium acetate, and 0.035 mg/ml sodium chloride) was used to obtain the baseline spectrum. Zarzio was not included due to lack of sample. Data were expressed as fluorescence intensity in function of temperature. All chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands).

Internalisation of nanodiscs and liposomes was quantified by fluorescence measurement using Rh-DHPE-labelled lipid nanodiscs. HeLa cells were plated at a density of approximately 20 × 10⁵ cells per mL with 1 mL of culture medium in 12-well plates (Costar 3513, Corning). A 10 mM stock solution of lipid nanodiscs and liposomes with 1% Rh-DHPE was prepared and diluted with PBS to the desired concentration. The final concentration of lipid (DPPC) was set to 1.56 μmol L⁻¹ in the culture medium with HeLa cells. PBS was used as a negative control. After the exposure of HeLa cells to lipid nanodiscs or liposomes for 1 hour at 37 °C in 5% CO₂ atmosphere, the cells were washed 3 times with 1 mL of PBS to remove excess nanodiscs or liposomes. To obtain the suspension of cells, the adhered HeLa cells were trypsinized. Collected cells were centrifuged at 1500 rpm for 3 minutes and washed twice with PBS at 37 °C. Finally, the cells were resuspended in 1 mL PBS and the number of cells was counted using a Neubauer-improved cell counting chamber. The fluorescence intensity of cellular suspension was measured using a JASCO FP-8300 fluorescence spectrometer (Tokyo, Japan). Emission intensity at 585 nm was recorded with the excitation at 560 nm. Excitation and emission band-passes were set to 10 nm. The fluorescence intensity was normalised against 10⁴ cells.

Experiments were performed in a JASCO FP-8300 Fluorescence Spectrometer with a PCT-818 Peltier Temperature Controller controlled by the Spectra Manager 2.0 (JASCO Deutschland GmbH, Pfungstadt, Germany). For each sample, 100 μL LNP suspension was loaded into a quartz microcuvette and fluorescence spectra was recorded at an excitation wavelength of 590 ± 20 nm and the fluorescence emission was recorded at a wavelength between 620 – 725 nm, with a 10 nm bandgap, 1 nm step, 0.2 s integration time, using high sensitivity, with 4 accumulations per scan. The emission spectra were recorded as the temperature ramped from 20°C to 90 °C, at a ramp rate of 2 °C / min, with scans taken every 10 °C.