HT-1080 untreated and PA supernatant treated cell pellet was resuspended in 0.1 M Tris (pH 8.0) and 150 mM NaCl containing protease/phosphatase inhibitor (1X) and sonicated (3X, 10s on 15s off, at 20%). After sonication, cell lysate was centrifuged at 12000 rpm for 14 min and the supernatant was collected and estimated for protein concentration using BCA kit. Reaction was performed using 120 μg of protein in buffer containing 0.1 M Tris (pH8.0), 1.25% triton X-100 along with 50 μM of HpETE-PE, glutathione (3 mM), glutathione reductase (1 U ml−1) and NADPH (120 μM). Consumption of NADPH was measured by fluorescence using Shimadzu spectrofluorimeter RF-5301 PC. Fluorescence measurements were performed in a 50 μl quartz cuvette using an excitation wavelength of 340 nm, emission wavelength 460 nm (excitation and emission slits of 5 nm and 3 nm, respectively).
Rf 5301 pc spectrofluorimeter
Manufactured by Shimadzu
Sourced in Japan
The RF-5301 PC spectrofluorimeter is a compact and versatile fluorescence spectrometer. It is designed to measure the fluorescence emission and excitation spectra of samples. The instrument features a stable xenon lamp as the excitation source, a monochromator for wavelength selection, and a photomultiplier tube as the detector. The RF-5301 PC is controlled by a personal computer and includes software for data acquisition and analysis.
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Lab products found in correlation
Cary 60 uv vis spectrophotometer, by Agilent Technologies (2 mentions)
Fura 2 am, by Thermo Fisher Scientific (2 mentions)
Lambda 365 spectrophotometer, by PerkinElmer (1 mentions)
Uv 3600 uv vis nir spectrometer, by Shimadzu (1 mentions)
Axima cfr plus, by Shimadzu (1 mentions)
Luna rp si c18 column, by Phenomenex (1 mentions)
Hplc system, by Gilson (1 mentions)
Minipuls 3, by Gilson (1 mentions)
Fls920 spectrofluorimeter, by Edinburgh Instruments (1 mentions)
3600 uv vis near infrared spectrophotometer, by Shimadzu (1 mentions)
240 elemental analyzer, by PerkinElmer (1 mentions)
Zetasizer nano zs 90 particle size analyzer, by Malvern Panalytical (1 mentions)
Spectrum one spectrophotometer, by PerkinElmer (1 mentions)
6520 q tof lc ms mass spectrometer, by Agilent Technologies (1 mentions)
Pti qm 400, by Horiba (1 mentions)
Quartz cuvette, by Hellma (1 mentions)
Palladium tetraphenyltetrabenzoporphyrin 1, by Frontier Scientific (1 mentions)
1 2 dimyristoyl sn glycero 3 phosphocholine, by Lipoid (1 mentions)
Sodium n carbonyl methoxypolyethylene glycol 2000 1 2 distearoyl sn glycero 3 phosphoethanolamine dspe mpeg 2000, by Lipoid (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
33 protocols using rf 5301 pc spectrofluorimeter
1
Quantifying HPETE-PE Consumption by Fluorescence
2
UV-vis and Fluorescence Spectroscopy
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UV-vis spectra of solutions were recorded on a PerkinElmer LAMBDA-365 spectrophotometer. Fluorescence emission spectra were measured by a Shimadzu spectrofluorimeter RF-5301PC. Solutions were placed in 1 cm path length quartz cells.
3
Curcumin I Permeabilizes Bacterial Cells
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PI intercalates with bases of deoxyribonucleic acid (DNA) and gives fluorescence. PI can enter the bacterial cell membrane only when it has been permeabilized through an agent, and bind with DNA. This DNA-bound PI fluoresces with excitation and emission at 544 nm and 620 nm, respectively. The fluorescence emission can be detected by both spectrofluorimeter [19 (link)] and flow cytometer [21 (link)] equipped with an appropriate filter. Briefly, S. aureus and E. coli cells were grown in BHI broth up to the mid logarithmic phase, harvested, washed, and adjusted to 106 CFU/ml in PBS buffer. The cells were incubated at 37°C with curcumin I (25 μM, 50 μM & 100 μM) and simultaneously with two bacterial membrane permeabilizing agents HNP-1 and nisin (both have been used as positive controls) at indicated time points. After curcumin exposure, the cells were washed in PBS buffer and incubated with PI (1.3 μg/ml) at 37°C for 20 min in dark. The PI fluorescence was measured at excitation and emission of 544 nm and 620 nm respectively, through a fluorescence spectrophotometer (Shimadzu RF-5301 PC spectrofluorimeter, Japan). Similarly, separate aliquots were prepared and measured in the flow cytometer (Becton Dickinson (BD) Facs verse, San Jose, CA, and Beckman Coulter, CA). A total of 10,000 cells were acquired for each flow cytometry analysis.
4
Characterization of Photochemical Compounds
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All chemical reagents, unless otherwise specified, were purchased from Sigma-Aldrich Co. All solvents were of reagent grade and were purchased from local companies. All solvents were dried and distilled prior to use by following standard procedures. 1H NMR spectra were recorded on a Bruker 500 MHz FT-NMR (Advance-DPX 300) spectrometer at 25 °C. The chemical shift (δ) and coupling constant (J) values were given in parts per million (ppm) and hertz (Hz), respectively. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra were obtained on an AXIMA-CFR PLUS (SHIMADZU) spectrometer using α-cyano-4-hydroxycinnamic acid as a matrix. High-resolution mass spectra (HRMS-FAB) were recorded on JEOL JM AX 505 HA mass spectrometer. UV/Vis spectra were obtained by using a Shimadzu UV-3600 UV/Vis-NIR spectrometer. Steady-state emission spectra at room temperature were obtained using a Shimadzu RF-5301PC spectrofluorimeter. In vivo fluorescence imaging studies were performed using an IVIS lumina II preclinical imaging system and analyzed using the IVIS Living Imaging 4.4 software (PerkinElmer Inc., Alameda, CA, USA). Photoacoustic imaging studies of the tissue mimicking phantom and live mice were performed using the MSOT system (MSOT inSight 64, iThera Medical GmbH).
5
Characterization of Fluorescent Nanoparticles
6
Fluorescence and HPLC Analysis Method
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A Shimadzu RF-5301PC spectrofluorimeter (Shimadzu Corporation, Analytical Instrument Division, Kyoto, Japan), equipped with a Xenon discharge lamp and quartz cells were used for molecular fluorescence measurements.
Solutions were propelled by Gilson Minipuls 3 peristaltic pump with PVC pumping tubes and 3 valves were used for FIA configuration.
All HPLC experiments were carried out on a Gilson HPLC system equipped with a Gilson 322 controller pump operating at 1.0 mL min−1, a Rheodyne 7725i injector with a 50 μL sample loop, and a variable-wavelength UV-Vis 156 Gilson detector measuring at 245 nm. The analytical column used was a Phenomenex Luna RP Si C18 column (5 μm, 100 Å, 250 × 3 mm) (Torrance, CA, USA), the separation was carried out at room temperature. All calculations were performed using Matlab 6.0 software. The surface responses were graphed using Statistica 6.0 software.
A pH meter (Orion Expandable Ion Analyzer, Orion Research, Cambridge, MA, USA) Model EA940 with combined glass electrode was used for monitoring pH adjustment.
Solutions were propelled by Gilson Minipuls 3 peristaltic pump with PVC pumping tubes and 3 valves were used for FIA configuration.
All HPLC experiments were carried out on a Gilson HPLC system equipped with a Gilson 322 controller pump operating at 1.0 mL min−1, a Rheodyne 7725i injector with a 50 μL sample loop, and a variable-wavelength UV-Vis 156 Gilson detector measuring at 245 nm. The analytical column used was a Phenomenex Luna RP Si C18 column (5 μm, 100 Å, 250 × 3 mm) (Torrance, CA, USA), the separation was carried out at room temperature. All calculations were performed using Matlab 6.0 software. The surface responses were graphed using Statistica 6.0 software.
A pH meter (Orion Expandable Ion Analyzer, Orion Research, Cambridge, MA, USA) Model EA940 with combined glass electrode was used for monitoring pH adjustment.
7
Characterization of Quantum Dot Properties
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UV–vis absorption spectra were recorded with a Shimadzu 3600 UV–vis near-infrared spectrophotometer. PL measurements were performed with a Shimadzu RF-5301 PC spectrofluorimeter. The excitation wavelength was 350 nm with a 3/3 nm slit. PL decay results were measured by an Edinburgh FLS 920 spectrofluorimeter or Femtosecond Laser as excitation resource and oscilloscope as signal receiver. All optical measurements were performed at room temperature under ambient conditions. TEM images were recorded by a Tecnai F20 electron microscope with an acceleration voltage of 200 kV. XRD result was carried out by using the D/max-2500/PC. ICP results were measured by a PE5300DV ICP equipment. Purified QDs powder was used in ICP and XRD measurements. QD powder was obtained by precipitating QD from solution with equal volume of isopropanol and followed by drying in vacuum. We utilized a commercial Ti:sapphire regenerative amplifier (Libra, Coherent) at 800 nm (1.55 eV) with a repetition rate of 1 kHz and pulse duration of ~90 fs to carry out the TA experiments. For broadband measurements, a second-harmonic light source at 3.1 eV was used as the pump beam and an optical parametric amplifier (OperA solo, Coherent) was used to provide a probe beam with tunable wavelength. The relative polarizations of the pump and probe beams were set to be at the magic angle (54.7°).
8
Fluorescence Spectroscopy Protocol
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Fluorescence intensities were measured using a Shimadzu RF-5301 PC spectrofluorimeter (Kyoto, Japan) outfitted with a xenon lamp (150 W) and 1 cm quartz cells. Slits of 5 mm were used to fix the excitation and emission monochromators. For heating, an electrical thermostatic water bath with a temperature range of 35–100 °C was utilized. The pH values of the buffer solutions were measured using the Jenway instrument pH-meter (combined electrode).
9
Comprehensive Characterization of Nanomaterials
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The 1H-nuclear magnetic resonance (1H-NMR) spectra were recorded in DMSO-d6 on a Bruker Spectrospin Avance 400 MHz NMR spectrophotometer. Chemical shifts are given in ppm downfield from tetramethylsilane. Infrared (IR) spectra were measured on a Perkin Elmer Spectrum One spectrophotometer. UV-vis absorption spectra were recorded on a Cary 60 UV-vis spectrophotometer (Agilent Technologies). High resolution mass spectrometry (HRMS) was carried out using an Agilent 6520 Q-TOF LC/MS mass spectrometer. Elemental analysis was performed on a Perkin-Elmer 240 elemental analyzer. Fluorescence spectra and resonance light scattering (RLS) spectra were measured at room temperature using a SHIMADZU RF-5301PC spectrofluorimeter. The transmission electron micrograph (TEM) images were recorded on a German Leica TCS-SP8 transmission electron microscope. A Malvern Zetasizer Nano ZS90 particle size analyzer was used for dynamic light scattering (DLS) studies.
10
Spectrophotometric and Fluorescence Characterization
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A JASCO V660 spectrophotometer (Hachioji, Japan)
was used to carry
out the absorption study, and the instrument was attached with a thermo
electrical controlling cell holder. The experiments were conducted
in quartz cuvettes (Hellma, Germany) of 1 cm path length at (293 ±
0.5) K. The titration procedures are described in detail previously.33 (link)−35 (link)Steady-state fluorescence titrations were conducted either
in PTI QM-400 (HORIBA Canada) or using a RF5301-PC spectrofluorimeter
(Shimadzu, Kyoto, Japan) in fluorescence-free quartz cuvette (1 cm)
as described.33 (link)−35 (link),52 (link) All titrations were
executed setting excitation and emission band passes of 5 and 5 nm,
respectively. For synchronous fluorescence, the excitation range was
220–340 nm and Δλ was set at 15 and 60 nm, respectively.
Lifetime measurements were performed using PTI QM-400 instrument.
was used to carry
out the absorption study, and the instrument was attached with a thermo
electrical controlling cell holder. The experiments were conducted
in quartz cuvettes (Hellma, Germany) of 1 cm path length at (293 ±
0.5) K. The titration procedures are described in detail previously.33 (link)−35 (link)Steady-state fluorescence titrations were conducted either
in PTI QM-400 (HORIBA Canada) or using a RF5301-PC spectrofluorimeter
(Shimadzu, Kyoto, Japan) in fluorescence-free quartz cuvette (1 cm)
as described.33 (link)−35 (link),52 (link) All titrations were
executed setting excitation and emission band passes of 5 and 5 nm,
respectively. For synchronous fluorescence, the excitation range was
220–340 nm and Δλ was set at 15 and 60 nm, respectively.
Lifetime measurements were performed using PTI QM-400 instrument.
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