Light-driven proton-pumping activity was measured according to the method described by Yoshizawa et al. (2012) (link), with the following modifications. Cells in approximately 120–160 mL of modified NSY medium were collected by centrifugation at 7,000×g at 4°C for 8 min. Cells were then washed three times with 100 mM NaCl and resuspended in 6 mL of 100 mM NaCl. The light source was a 300-W xenon lamp (MAX-303; Asahi Spectra), and 6 mL of the cell suspension was initially placed in the dark and then irradiated for 5 min through 450-±10-nm, 520-±10-nm, and 580-±10-nm bandpass filters (MX0450, MX0520, and MX0580; Asahi Spectra). The light intensity of each wavelength was fixed at approximately 7 mW cm–2 using an optical power meter (#3664; Hioki) with an optical sensor (#9742; Hioki). pH was measured using a pH meter (LAQUA F-72; Horiba). Proton-pumping activity was defined as the slope in the 30 s following the onset of illumination. This initial slope was normalized to that obtained prior to illumination. The first measurement was performed without external retinal, and the second measurement was conducted after the addition of retinal dissolved in 100% ethanol (at a final concentration of 20 μM). All measurements were performed at 4°C.
Laqua f 72
Manufactured by Horiba
Sourced in Japan
The LAQUA F-72 is a portable water quality meter designed for field measurement of pH, ORP, and temperature. It features a waterproof and dust-proof casing, easy-to-read LCD display, and automatic temperature compensation. The meter is suitable for a variety of applications that require on-site water quality analysis.
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Lab products found in correlation
Ssm 5000a, by Shimadzu (3 mentions)
Millex gv, by Merck Group (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Vacutainer, by BD (1 mentions)
Vacutainer tube, by BD (1 mentions)
Gmrd 103, by Analox Instruments (1 mentions)
9618s 10d micro touph electrode, by Horiba (1 mentions)
Gm7 microstat, by Analox Instruments (1 mentions)
15 protocols using laqua f 72
1
Measuring Light-Driven Proton Pumping
2
Purification of Recombinant α-Synuclein
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Recombinant human α-synuclein (αSN) was expressed in Escherichia coli and purified as described previously (Yagi et al., 2005 (link), Yagi et al., 2010 (link)). In all experiments, lyophilized αSN was dissolved in MilliQ water and filtered by Millex-GV with a pore size of 0.22 μm (Merck, Tokyo, Japan). Then, a PD-10 column (GE Healthcare Japan Co., Tokyo, Japan) was used for the removal of residual salt prior to the preparation of all monomeric samples. After the removal of salt, the concentration of αSN was measured spectrophotometrically using a molar extinction coefficient of 5120 M−1cm−1 at 280 nm. The pH of all samples was adjusted to pH 4.7 by sodium phosphate buffer with an error of less than 0.1 by pH meter LAQUA F-72 (HORIBA Ltd., Kyoto, Japan).
ThT was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were obtained from Nacalai Tesque, Inc. (Kyoto, Japan).
ThT was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were obtained from Nacalai Tesque, Inc. (Kyoto, Japan).
3
Fecal pH and SCFA Quantification
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The pH and SCFA concentrations of fecal samples were measured by the following method.
Approximately 3 g of fecal samples were transferred to sterile tubes, mixed with 6
ml of distilled water (1:2 dilution ratio), and vortexed for 5 min.
Each sample was centrifuged (10,000 × g for 5 min at 4°C), and
supernatant was transferred into a sterile microtube to measure SCFAs. The pH of the
residual supernatant was measured using a pH meter (LAQUA F-72, Horiba, Kyoto, Japan). The
microtubes for SCFA quantification were centrifuged (16,000 × g for 5 min
at 4°C) again, and the supernatant was filtered through a 0.45 µl filter
(Sartorius, Goettingen, Germany). Filtrated samples were subjected to high-performance
liquid chromatography (Shimadzu Corp., Kyoto, Japan) to measure SCFA concentrations.
Approximately 3 g of fecal samples were transferred to sterile tubes, mixed with 6
ml of distilled water (1:2 dilution ratio), and vortexed for 5 min.
Each sample was centrifuged (10,000 × g for 5 min at 4°C), and
supernatant was transferred into a sterile microtube to measure SCFAs. The pH of the
residual supernatant was measured using a pH meter (LAQUA F-72, Horiba, Kyoto, Japan). The
microtubes for SCFA quantification were centrifuged (16,000 × g for 5 min
at 4°C) again, and the supernatant was filtered through a 0.45 µl filter
(Sartorius, Goettingen, Germany). Filtrated samples were subjected to high-performance
liquid chromatography (Shimadzu Corp., Kyoto, Japan) to measure SCFA concentrations.
4
Rhodopsin Heterologous Expression and Characterization
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All rhodopsin genes used in heterologous expression studies were codon optimized and chemically synthesized by Eurofins Genomics (Tokyo, Japan, Supplemental Table 1 and 2 ). These fragment DNAs were inserted into NdeI and XhoI sites of pET21a vector (Novagen, Madison, WI, USA) to express the rhodopsin in E. coli C41(DE3, Lucigen). All rhodopsins were overexpressed in cells grown at 37 °C in 200 ml of 2xYT medium and induced at an optical density (600 nm) of 0.4–0.6 with 0.1 mM IPTG and 10 μM all-trans-retinal. The rhodopsin-expressed cell suspension then was placed in darkness and illuminated using a 300 W xenon lamp (MAX-303; Asahi Spectra, Tokyo, Japan) for 3 min. Light-induced pH changes were measured using a pH meter (LAQUA F-72; Horiba, Kyoto, Japan). The measurements were then repeated under the same conditions after the addition of the protonophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP, final concentration, 10–30 μM). All measurements were performed in 100 mM NaCl at 4 °C.
5
Measuring Mouse Brain pH
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Mice were killed via cervical dislocation and decapitation, following which whole brains were removed. The brains were immediately frozen in liquid nitrogen and stored at –80 °C until use. We measured brain pH as previously described (Catts et al, 2005 (link); Halim et al, 2008 (link)). Briefly, mouse brains were homogenized using a tissue homogenizer equipped with a conical pestle in ice-cold distilled H2O (5 ml per 500 mg of tissue). The pH was measured using a pH meter (LAQUA F-72, Horiba Scientific, Kyoto, Japan) after a three-point calibration at pH 4.0, pH 7.0, and pH 9.0. The pH experiments were performed in triplicate for each sample, following which homogenates were immediately frozen and stored at –80 °C until required for further analyses.
6
Soil Nutrient Extraction and Analysis
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Soil pH (1:2.5 soil-to-water suspension, w/v) was analyzed using a pH meter (LAQUA F-72, Horiba, Kyoto, Japan). The diethylenetriaminepentaacetic acid (DTPA) method was used to extract the Fe concentration from soil [14 (link)]. Similarly, Ca was extracted by the ammonium acetate method [15 (link)], while Fe and Ca concentrations in the extracts were measured using an atomic absorption spectrophotometer (Hitachi Z2300, Hitachi High-Technologies Corporation, Tokyo, Japan). Finally, the Al concentration in soil was estimated using a titrimetric method after extracting the soil sample with KCl [16 ]. The forms of Fe, Ca, and Al were named “available Fe”, “exchangeable Al”, and “exchangeable Ca” according to the previous reports [15 (link),17 (link),18 (link)].
7
Amyloid Fibril Formation Kinetics
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The sample solutions of 200 μL containing 0.3, 0.1, or 0.03 mg/mL of αSN, 0–500 mM NaPi (pH 2–9), and 5 μM ThT were prepared in each well of a 96-well microplate at 37 °C. Five wells were used for each solution condition to confirm reproducibility. pH values of samples were confirmed by a pH meter (LAQUA F-72, HORIBA, Kyoto, Japan) before fibril formation.
ThT fluorescence was measured using the Handai Amyloid Burst Inducer (HANABI) (CORONA ELECTRIC, Ibaraki, Japan), in which a microplate reader was combined with a water bath-type ultrasonicator, with excitation and emission wavelengths at 445 and 485 nm, respectively (Umemoto et al., 2014 (link)). Ultrasonication was applied to accelerate amyloid formation with a cycle of 1-min irradiation and 9-min quiescence. The lag time was calculated as the time at which the kinetics of the ThT assay reached 1/10 of the maximum intensity.
ThT fluorescence was measured using the Handai Amyloid Burst Inducer (HANABI) (CORONA ELECTRIC, Ibaraki, Japan), in which a microplate reader was combined with a water bath-type ultrasonicator, with excitation and emission wavelengths at 445 and 485 nm, respectively (Umemoto et al., 2014 (link)). Ultrasonication was applied to accelerate amyloid formation with a cycle of 1-min irradiation and 9-min quiescence. The lag time was calculated as the time at which the kinetics of the ThT assay reached 1/10 of the maximum intensity.
8
Comprehensive in vitro digestibility analysis
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After 24 h of incubation, the total gas production was measured, and gas samples were collected from the headspace of the glass bottles into vacutainer tubes (BD Vacutainer®, Becton Drive, Franklin Lakes, NJ, USA). The tubes were stored at room temperature until CH4 and CO2 determination. Thereafter, the bottles’ caps were removed, and the pH of each tube was recorded using a pH meter (LAQUA F-72, HORIBA Scientific, Kyoto, Japan). Then, aliquots of the culture fluid were transferred into 1.5 mL Eppendorf tubes and centrifuged at 16,000× g and 4 °C for 5 min. The supernatant was collected and transferred into a new Eppendorf tube® (Eppendorf AG, Hamburg, Germany), which was stored at −20 °C until use for volatile fatty acid (VFA) and ammonia nitrogen (NH3-N) analysis. The bags were removed from the bottles, washed under running tap water until the draining fluid became clear, and then dried at 60 °C for 48 h to determine the In Vitro dry matter digestibility (IVDMD). After IVDMD determination, the bags were used for the determination of In Vitro organic matter digestibility (IVOMD), In Vitro neutral detergent fiber digestibility (IVNDFD), and In Vitro acid detergent fiber digestibility (IVADFD). The residues in the fermentation bottles were discarded.
9
pH Measurement of Plant Extracts
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The pH values of the EWS, CIS, YPF, and BBL extracts were gauged using a pH meter (LAQUA F-72, Horiba, Ltd., Kyoto, Japan). Each treatment group underwent five measurements.
10
In Vitro Rumen Fermentation Analysis
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After 24 h of incubation, the total gas production was estimated, and a sample of the headspace gas was collected from each bottle and stored in a vacutainer tube (BD, Becton Drive, NJ, USA), which was stored at room temperature until CH4 and CO2 determination. Afterward, the fermentation bottle caps were removed, and the pH was immediately determined (LAQUA F-72, HORIBA Scientific, Kyoto, Japan). Approximately 1 mL of the culture media was collected into Eppendorf tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 16,000× g and 4 °C for 5 min. The supernatant was collected and stored at −20 °C until use for volatile fatty acids (VFA) and ammonia nitrogen (NH3-N) estimation. The filter bags were washed with tap water until the effluent was clear. Then, the bags were dried at 60 °C for 48 h to estimate the in vitro dry matter digestibility (IVDMD). Afterwards, the bags were used for estimation of the in vitro organic matter digestibility (IVOMD), in vitro neutral detergent fibre digestibility (IVNDFD), and in vitro acid detergent fibre digestibility (IVADFD).
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