Naive CD4+ T cells isolated from PB of healthy donors were suspended at 3 × 106 cells/ml in Hank's balanced salt solution (HBSS) containing 1 mg/ml of bovine serum albumin (BSA) and 10 mM HEPES, pH 7.4 (HBSS-BSA) and incubated with 1 mM Fura-2-AM (Dojindo, Kumamoto, Japan) at RT for 30 min in the dark. After washing twice with HBSS-BSA, cells were suspended in HBSS-BSA at 2.5 × 106 cells/ml. 2 ml of the cell suspension in a quartz cuvette was placed in a luminescence spectrometer (RF-5000, Shimadzu, Kyoto, Japan) and fluorescence was monitored at an emission wavelength of 510 nm, and excitation wavelengths of 340 and 380 nm every 20 ms. Calibration of fluorescence in terms of [Ca2+]i was calculated from the ratio 340/380 excitation fluorescent values.
Rf 5000
Manufactured by Shimadzu
Sourced in Japan
The RF-5000 is a fluorescence spectrophotometer manufactured by Shimadzu. It is designed to measure the fluorescence emission of samples.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Fura 2 am, by Dojindo Laboratories (1 mentions)
Glycogen assay kit, by Abcam (1 mentions)
Digitonin, by Merck Group (1 mentions)
Infinite 200m, by Tecan (1 mentions)
Anti biotin microbead, by Miltenyi Biotec (1 mentions)
Fura 2 am, by Beyotime (1 mentions)
Fura 3 am, by Beyotime (1 mentions)
14 protocols using rf 5000
1
Fura-2-AM Intracellular Calcium Measurement
2
Liver Cholesterol, Triglyceride, and Glycogen Quantification
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Liver homogenates were prepared in a tenfold volume v/w of isopropyl alcohol and then supernatants were collected after centrifugation at 12 000 × g and 4 °C for 15 min. Liver cholesterol content was examined as previously described.[12 , 35 ] In brief, liver tissues were washed with phosphate‐buffered saline (PBS) and harvested in isopropyl alcohol followed by ultrasonication. Sonicates (0.1 mL) were added to assay solutions (0.9 mL); 0.1 U mL−1 cholesterol oxidase, 0.01 U mL−1 cholesterol ester hydrolase, 1 U mL−1 peroxidase, 0.05% Triton X‐100, 1 mmol L−1 sodium cholate, and 0.6 mg mL−1 β‐hydroxyphenylacetic acid; pH 7.4) and incubated at 37 °C for 1 h. Fluorescence of the mixture was detected by a fluorospectrometer (RF‐5000; Shimadzu Co., Tokyo, Japan). Triglycerides were determined using the Triglyceride Kit (Wako Diagnostics, Richmond, VA). Total glycogen level was measured with the Glycogen Assay Kit (BioVision, Irvine, CA) according to the manufacturer's instructions. Hepatic cholesterol, triglycerides, and glycogen were normalized to the amount of protein.
3
Measuring Intracellular pH using pHluorin
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Strains transformed with the plasmid pYES-PACT1-pHluorin were grown to an OD600 of 0.5–0.75 in SC medium, centrifuged and resuspended in PBS containing 0.1 M digitonin (Sigma), which permeabilizes cell membranes. After 10 min, cells were washed with PBS at 4 °C and resuspended in citric acid/Na2HPO4 buffer of pH values ranging from 5.0 to 8.0. Cell fluorescence was measured using a fluorimeter (Shimadzu RF-5000). pHluorin fluorescence emission was measured at 508 nm providing excitation bands of 395 nm and 475 nm [34 (link),35 (link)]. A calibration curve was generated plotting the ratio of emission intensity resulting from excitation at 395 and 475 nm (395/415 ratio) against the corresponding buffer pH [35 (link)]. Background fluorescence for a WT culture not expressing pHluorin was subtracted from the measurements. All pH determination experiments were repeated, at least, three times (biological replicates). To analyze changes in intracellular pH upon acetic acid stress, strains were grown to an OD600 of 0.5 in SC medium, centrifuged and resuspended in SC medium plus the indicated amount of acetic acid (pH adjusted to 4.5). After 5 and 10 min, fluorescence emission (395/415 ratio) was measured and converted to intracellular pH according to the calibration curve previously obtained.
4
Synthesis and Characterization of HA-Tyramine Conjugate
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The HA-Tyr were synthesized according to the reported method by amide bond formation via carbodiimide-mediated condensation of the carboxyl groups of HA with the amino group of tyramine using WSCD and NHS in MES buffer [35 (link)–38 (link)]. The synthesized HA-Tyr was characterized by 1H-NMR spectroscopy in D2O at 30 °C, and the degree of substitution of the Ph residue in the substrates was determined by UV–Vis spectrophotometer (Shimadzu RF -5000) at 275 nm using a calibration curve established for known percentages of tyramine hydrochloride. The HA-Tyr was sterilized with 96% ethanol solution under a laminar flow hood and dissolved in phosphate buffer saline (PBS, pH = 7.4). The HRP and other chemicals were sterilized using 0.2 µm pore membrane filters to remove microbial contamination. The microfluidic device, connecting tubes, liquid paraffin, and lecithin were sterilized using a steam autoclave. In addition, adipose-derived stem cells (ADSCs) from rat tissue were cultured as model cells in Dulbecco’s modified Eagle’s medium (Gibco Technologies, Logan, UT) containing 10% (v/v) heat-inactivated fetal bovine serum, 100 µg/mL penicillin and streptomycin under physiological conditions.
5
In vitro Toxicology of Nanoparticles on Human Cells
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Effects of nanoparticles on the mononuclear human cells were examined by various biological assays. Fresh, healthy and untreated bloods of 15 donors were utilized for these studies. Peripheral blood (PB) samples were obtained after informed consent on age-matched controls (20 to 25 years), and health assurance was confirmed by clinical examination as well as morphological and immunological criteria. The study was approved by the research ethics committee of the Shahid Beheshti University of Medical Sciences (Tehran, Iran) and all the patients signed an informed consent form. Instruments utilized for the in vitro toxicology investigations include a MCO 17A1 CO2 incubator (Sensor Sanyo IR, Japan), vapor bath stark eliwellewpc 800 (UK), Harrier 18/80 refrigerated centrifugation (Sanyo, Japan), UV/Visible spectrometer (Shimadzu 160 ABB, Japan), floremetry (Shimadzu RF-5000, Japan), digital balance (Shimadzu 20 E8 330 H, Japan), shaker (REAX2000, Iran), ELISA reader (In finite 200 M, TECAN, Rainbow Thermo, Austria), and BD Biosciences FACS Calibure TM flow cytometer equipped with a 488 nm argon ion laser and a 530 nm band pass filter (FL-1 channel). Details of experimental analyses are explained below.
6
Spectrophotofluorometric Quantification of Neurotransmitters
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Concentrations of NE, DA, and 5-HT were determined in the hippocampal tissue by the spectrophotofluorometric method (Shimadzu RF-5000). Transmitter’s oxidations were done by adding 0.1 N iodine followed by stopping the oxidation by alkaline sulfite addition to produce specific fluorescence. The induced fluorescence was quantified at a particular emission wavelength following the excitation at another specific wavelength that varies according to the transmitter. The intensity of the fluorescence is directly proportional to the neurotransmitter level in the brain tissue (Aziz et al., 2013 (link)).
7
Fura-2 AM-based Calcium Imaging in Rat Cardiomyocytes
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Ventricular cardiomyocytes were isolated from the rat hearts using Saini et al.'s method.16 (link) Then, the isolated ventricular cardiomyocytes were treated in a fura-2 AM buffer (5 μM fura-2 AM with 1 mM Ca2+, pH 7.4) for 40 minutes. The cells were then rinsed twice to remove excess dye. For standardization, the cell density within the cuvette was set to 300,000 cells/mL per experimental group. Fluorescent intensities were measured with a dual-wavelength spectrofluorometer (RF-5000, Shimadzu Corporation, Kyoto, Japan) using the following parameters: 340/380-nm excitation wavelength, 510-nm emission wavelength, 0.95-s integration time, and 1.0-s resolution time. [Ca2+]i levels were assessed with the Grynkiewicz formula as follows17 (link): [Ca2+]i = Kd × [(R − Rmin)/(Rmax − R) × Sf2/Sb2], where R is the fluorescence signal ratio derived from 340-nm and 380-nm excitation wavelengths, Rmax and Rmin are the R values after adding 10% Triton X-100 (20 μL) and 400 mM EGTA (40 μL), respectively, and Sf2 and Sb2 are the fluorescence proportionality coefficients (measured at a 380-nm excitation wavelength) obtained under Rmin and Rmax conditions, respectively.
8
Thioflavin T Fluorescence Assay for Amyloid Fibrillogenesis
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Thioflavin T (ThT) fluorescence assay is frequently used to evaluate the fibrillogenesis (amyloids) rate. A ThT molecule produces fluorescence upon binding to amyloids and could be used to monitor in vitro amyloid fibril aggregation (Xue et al., 2017 (link)). The leaves extracts (10–100 μg/mL) were incubated with 10 µM of Aβ1-40 (40 µL) at 37°C for 1 day. Then, 100 µL of ThT (10 µM in PBS buffer, pH 7.4) was added. An FP-6200 spectrofluorometer (Shimadzu RF-5000) was used to assess the amounts of ThT fluorescence in the samples and the fluorescence was measured after 30 min at the wavelengths of excitation and emission Ex450nm and Ex483nm respectively along with the positive control (Galantamine + Aβ).
9
Measuring Intracellular Calcium Dynamics in CTLs
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Intracellular calcium fluxes were measured as described previously (21 (link)). Briefly, CTLs with ASM or CD161 silencing were labeled with Fura-2-AM (Beyotime, #S1052, 1 μmol/L) for 30 minutes at 37°C. For CD161 cross-linking, T cells were incubated with anti-biotin microbeads (Miltenyi Biotec, #130-092-357) loaded with biotinylated anti-CD161 antibody (Miltenyi Biotec, #130-092-906) or isotype-biotin control (BioLegend, # 400104) for 3 minutes at 37°C. Intracellular Ca2+ was recorded at 500 nm emission in response to 340 nm/380 nm excitation using a fluorescence spectrophotometer (RF-5000; Shimadzu). T cells were stimulated by the addition of 40 μg/mL biotinylated anti-CD3ε (Miltenyi Biotec, #130-093-377, clone OKT3) followed by 20 μg/mL streptavidin (Thermo Fisher Scientific, #434301). For flow-cytometric analysis, T cells were labeled with Fura-3-AM (Beyotime, #S1056, 1 mmol/L) and Live/Dead Fixable Viability Dye-eFluor780. Calcium fluorescence was monitored at an emission wavelength of 530 nm with excitation at 488 nm. For fluorescent microscopy imaging, cells were loaded with Furo-3-AM, Hoechst 33342 (Thermo Fisher Scientific, # 62249), and PE-conjugated anti-human CD8a antibody (BioLegend, #301008) on a confocal dish and analyzed on an LSM 780.
10
Recombinant ranibizumab interaction analysis
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The eGFP split reporter was used to study the recombinant ranibizumab heavy and light chain interaction. Two complementary segments of eGFP were fused via a linker to the recombinant heavy and light chains. To analyze the complementation of the segments, the equal amounts of supernatants of two yeast cultures producing heavy and light chains with complementary parts of eGFP were mixed and incubated for 24 hours at 4°C. In another experiment, the cited mixture was incubated with substrate VEGF-A. The supernatants of yeast cultures producing non-chimeric heavy and light chains and the mixture of them were evaluated as well. The empty BMGY medium and the supernatant of eGFP producing yeasts were negative and positive controls, respectively. The intensity of fluorescence was measured using the spectrofluorometer (Shimadzu RF-5000, Japan) with 470 nm excitation and 520 nm emission.
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