Interaction of IR and vanadium compound, dmp was also determined by the fluorescence titration experiment. Room temperature tryptophan fluorescence was measured in recombinant IR with the concentration of 0.3 μM in 50 mM Phosphate buffer pH7.4, 50mM NaCl using Hitachi spectrofluorometer with the slit width of 10 nm. The emission spectra were recorded from 300 to 500 nm with an excitation wavelength 280 nm. Fluorescence intensity of vanadium compound was negligible in this specific excitation wavelength. The fluorescence intensities of IR under different concentration at 346 nm were monitored. The fluorescence quenching constant was estimated from Stern Volmer plot analysis.
Spectrofluorometer
Manufactured by Hitachi
Sourced in Japan
The Hitachi Spectrofluorometer is a laboratory instrument used for the analysis and measurement of fluorescence in samples. It functions by exciting molecules in the sample with a beam of light and detecting the resulting fluorescent emission.
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16 protocols using spectrofluorometer
1
Fluorescence-based Insulin Receptor Interaction
2
Intracellular ROS Quantification with DCF-DA
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Intracellular ROS production was estimated by using 2, 7-dichlorofluorescein diacetate (DCF-DA) as a probe according to the method of Kuo and Tang (23 (link)). Briefly, 100 μl of cell lysate was incubated with the assay media (20 mM tris-HCl, 130 mM KCl, 5 mM MgCl2, 20 mm NaH2PO4, 30 mM glucose and 5 μM DCF-DA) at 37°C for 15 min. The formation of DCF was measured at the excitation wavelength of 488 nm and emission wavelength of 510 nm for 10 min by using spectro-fluorometer (Hitachi) equipped with a FITC filter.
3
Oxidative Stress Biomarkers in Liver
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The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) were measured using a commercially available kit (Nanjing Jiancheng bioengineering institute, Nanjing, China). Briefly, liver weights were measured and homogenized in 2 ml ice-PBS. The samples were centrifuged at 2000 g for 10 minutes at 4℃. The MDA levels in the supernatant was determined by thiobarbituric acid (TBA) reaction and the absorbance was measured at 532 nm. The GSH levels were measured by the enzymatic recycling method using glutathione reductase and 5', 5'-dithio-bis (2-nitrobenzoic acid) (DTNB). 2-nitro-5-thiobenzoic acid (TNB) formation is monitored at 412 nm. ROS levels in liver tissues were determined using 2, 7-dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe. The supernatant (190 µl) was incubated with 1 mM DCFH-DA (10 µl) at 37℃ for 30 min. The fluorescence intensity was measured at an excitation and emission wavelength of 485 nm and 525 nm, respectively, using a spectrofluorometer (Hitachi, Tokyo, Japan).
4
Fluorescence and Nitrite Assays
5
Measuring β-Glucosidase Activity in Plant Epidermis
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To measure β-glucosidase activity, the separated epidermis was pulverized using phosphate-buffered saline (1x, pH 7.2) supplemented with 100 µM phenylmethanesulfonyl fluoride (PMSF) and centrifuged at 10,000 × g for 5 min at 4°C. The separated supernatant was reacted with citrate-phosphate buffer (pH 5.6, 5 mM sodium taurocholate) containing 4-methylumbellifery-β-D- glucopyranoside (4-MUG) at 37°C for 60 min. The reaction was terminated by adding 200 mM carbonate-bicarbonate buffer (pH 10.5) and the fluorescence intensity of 4-methylumbelliferone (4-MU) converted from 4-MUG was measured using a spectrofluorometer (Hitachi, Ltd., Tokyo, Japan) at excitation and emission wavelengths of 360 and 450 nm, respectively. The 4-MU concentrations ranging from 0 to 300 nM were used as the standard for fluorescence measurements.
6
Evaluating Mitochondrial Membrane Fluidity
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Fluidity of mitochondrial membranes was evaluated by fluorescence anisotropy of mitochondria-bound dye DPH. DPH (20 mM in tetrahydrofuran) was first diluted 100 times with 10 mM Tris-HCl, 150 mM KCl, 1 mM EDTA, pH 7.4. Subsequently, DPH was injected into stirred mitochondrial suspensions (0.5 mg/ml) and the mixture was incubated for 30 min at 37 °C. Fluorescence polarization was measured in a Hitachi spectrofluorometer at wavelengths of 366 nm for excitation and 425 nm for emission. The results are expressed as anisotropy units (r), where r = (I0/I90)/(I0+2I90). I0 and I90 represent the intensities of light when polarizers were in parallel or perpendicular orientation, respectively. Light scattering and intrinsic fluorescence were routinely corrected by subtracting the signal obtained from unlabeled samples and the fluorescence of the buffer plus label alone.
7
Proteolytic Activity Assay Methods
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General proteolytic activity was assayed against acid hemoglobin according to Anson method with modifications (Szymczak 2017 (link)). The general proteolytic activity was defined as mg tyrosine liberated per 1000 g meat or 1000 mL brine at 37 °C within 2 h (UHb). The activity of D-like cathepsin was determined using 1 µM pepstatin-A (Sigma–Aldrich, Poland) and its percent inhibition was calculated. Analyses were performed in three replications.
Activities of aspartyl and cysteine peptidases were measured against Mca-GKPILFFRLK(Dnp)-r-NH2 and Z-FR-MCA (PeptaNova, Concord, CA, USA), respectively (Szymczak 2017 (link)). Fluorescence was measured with a Spectrofluorometer (Hitachi, F-7000, Tokyo, Japan) using microcuvette with excitation and emission wavelengths at 328 and 393 for aspartyl, and 322 and 460 nm for cysteine peptidases, respectively. One unit of enzyme activity (UMCA) was defined as 1 nmol MCA released from 1 g of meat or 1 mL of brine per minute at 37 °C. Analyses were performed in two replications.
Activities of aspartyl and cysteine peptidases were measured against Mca-GKPILFFRLK(Dnp)-r-NH2 and Z-FR-MCA (PeptaNova, Concord, CA, USA), respectively (Szymczak 2017 (link)). Fluorescence was measured with a Spectrofluorometer (Hitachi, F-7000, Tokyo, Japan) using microcuvette with excitation and emission wavelengths at 328 and 393 for aspartyl, and 322 and 460 nm for cysteine peptidases, respectively. One unit of enzyme activity (UMCA) was defined as 1 nmol MCA released from 1 g of meat or 1 mL of brine per minute at 37 °C. Analyses were performed in two replications.
8
Mitochondrial Membrane Fluidity Analysis
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Fluidity of mitochondrial membranes was evaluated by fluorescence anisotropy of mitochondria-bound dye DPH. DPH (20 mM in tetrahydrofuran) was first diluted 100 times with 10 mM Tris-HCl, 150 mM KCl, 1 mM EDTA, pH 7.4. Subsequently, DPH was injected into stirred mitochondrial suspensions (0.5 mg/ml) and the mixture was incubated for 30 min at 37 °C. Fluorescence polarization was measured in a Hitachi spectrofluorometer at wavelengths of 366 nm for excitation and 425 nm for emission. The results are expressed as anisotropy units (r), where r = (I0/I90)/(I0+2I90). I0 and I90 represent the intensities of light when polarizers were in parallel or perpendicular orientation, respectively. Light scattering and intrinsic fluorescence were routinely corrected by subtracting the signal obtained from unlabeled samples and the fluorescence of the buffer plus label alone.
9
Mitochondrial Superoxide Detection in HK-2 Cells
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The mitochondrial superoxide levels were detected using mitochondrial superoxide detection kit (Abcam 19943). The HK‐2 cells were allowed for overnight attachment and exposed for normoxia and hypoxia (6 and 16 hours) conditions. Followed by this, mitoROS working solution was added and incubated at 37℃ for 60 minutes. The fluorescence intensity was measured at 540/590 nm using fluorescence reader (Hitachi Spectrofluorometer).
10
Chlorophyll a Fluorescence at 77K
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Low-temperature Chl a fluorescence spectra at 77K were measured according to Cederstrand and Govindjee (1966) (link) using spectrofluorometer (Hitachi, Japan) and applying a monochromatic exciting light with wavelength of 435 nm.
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