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38 protocols using ultrospec 2100 pro

1

Yeast Cell Growth Rate Determination

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The growth rate of yeast cells were determined by measuring OD660 after appropriate dilutions using a spectrophotometer (Ultrospec 2100 Pro, GE healthcare, Fairfield, USA) every 8 h. The measurements were continued until the OD660 nm reached the plateau. This experiment was repeated 3 times. The growth curve of different strains was drawn by software of Excel Microsoft Office Professional Plus 2013 (Microsoft, Redmond, USA).
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2

Quantification of Tick H2O2 Levels

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The H2O2 concentration in ticks was measured using the ferrous oxidation of xylenol orange assay [18 (link)]. Briefly, homogenized unfed and partially fed ticks were suspended in 200 μl of Milli-Q H2O, while homogenized engorged ticks were suspended in 900 μl of Milli-Q H2O. The samples were centrifuged at 500× g, and the supernatant was collected. The supernatant from the engorged ticks was further diluted 10 times in Milli-Q H2O. Ninety microliters of the supernatant from unfed and partially fed ticks or the diluted supernatant from engorged ticks was used for a sample solution as described later. The assay reagent consisted of 125 μM xylenol orange, 250 μM ammonium iron (II) sulfate, 100 mM sorbitol, and 25 mM sulfuric acid. One hundred microliters of the sample solutions was added to a 1-ml assay reagent. The mixture was vortexed immediately, left at room temperature for 30 min, and measured at 560 nm using a spectrophotometer (Ultrospec 2100 pro; GE Healthcare, Pittsburgh, PA, USA). Finally, the ratio of the H2O2 concentration (μM) to the corresponding tick’s body weight (mg) was calculated.
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3

Quantifying β-Glucan in Microbial Cells

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The amount of β-glucan per gram cells were measured using aniline blue as described previously with some modifications [40 (link), 41 (link)]. Cells were grown to early log phase (2.5 × 106 cells) and harvested (10,000×g, 1 min). The cells were washed twice with 1 ml TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 8.0), resuspended to 250 μl in TE and then 6 M NaOH was added to a final concentration of 1 M. Following incubation in a water bath at 80 °C for 30 min, 1.05 ml of AB mix [0.03 % aniline blue (Sigma-Aldrich, USA), 0.18 M HCl, and 0.49 M glycine/NaOH, pH 9.5] was added. The tube was vortexed briefly and then incubated at 50 °C for 30 min. Fluorescence of β-glucan was quantified using a spectrofluorophotometer (Ultrospec 2100 Pro, GE healthcare, Fairfield, USA). Excitation wavelength was 400 nm and emission wavelength was 460 nm. This experiment was repeated three times.
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4

Yeast Growth Rate Measurement Protocol

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The yeast strains were inoculated into YPD medium and incubated at 30°C for 24 h (pre-culture). Cell growth was monitored by measuring the optical density of the cultures at 600 nm (OD600) using a spectrophotometer (Ultrospec 2100 pro; GE Healthcare, Freiburg, Germany). The cells were transferred to YPD medium at a starting OD600 of 0.1 and incubated at 30°C with shaking. The experiments were performed in triplicate. Growth rates, expressed as the time required for the cultures to double in optical density (doubling time, Td), were calculated by comparing the optical density at multiple time points during the linear growth phase and using the following formula: Td=t2t1/log2OD2/OD1.
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5

Quantitative Analysis of Protein-Gene Expression

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To further understand the relationship between proteins and their encoding genes, qPCR was run for proteins of differential hepatic abundance at the mRNA level. Specific primers for target genes of the identified proteins were designed using the primer BLAST of NCBI and nucleotide information in GenBank (Additional file 2: Table S2). Total RNA was prepared from the liver of control and treated groups using TRNzol-A+ (TIANGEN, Beijing, China). RNA quality and concentration were detected using spectrophotometer (Ultrospec 2100 pro, GE Healthcare) and agarose gel electrophoresis. cDNA synthesis with 5 μg of RNA was performed using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The PCR was performed in a 20-μL reaction system containing 1 μL of cDNA, 0.5 μL of each primer (10 μM), 10 μL of Super Real PreMix (SYBR Green) (TIANGEN) and 8.2 μL of water. The fold-change was calculated using the IQTM5 software (Bio-Rad) with the 2 −ΔΔCt method [25 (link)]. All operation for qPCR was followed by the MIQE [26 (link)].
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6

Chlorophyll and Xanthophyll Analysis

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Chl a/b ratios and total Chl content were estimated by absorption spectra (Ultrospec 2100 pro spectrophotometer, GE Healthcare) in a final 80% acetone concentration. Leaf material extraction was performed according to Porra et al. (1989) (link). Xanthophyll concentrations were determined using reverse-phase HPLC, in 100% methanol, with a LiChrospher 100 RP-18 column and Dionex Summit chromatography system (Ruban et al., 1994 (link)). Xanthophyll proportions were calculated as [mmol of a xanthophyll specie/(mmol of total xanthophylls))]. Intact chloroplasts were used for xanthophyll determination. Chloroplasts extraction and HPLC analyses were performed on three plants from each genotype, with chloroplast intactness measured by Fv/Fm.
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7

Ferric Reducing Antioxidant Potential Assay

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Antioxidant potential was measured using the ferric reducing ability of plasma (FRAP) assay according to the procedure described by Benzie and Strain [57 (link)], with minor modifications. Briefly, 100 μl of 1% (v/v) test sample was mixed with 900 μl of freshly prepared FRAP reagent, consisting of 20 mM ferric chloride (in water), 10 mM TPTZ (in 40 mM HCl), and 300 mM sodium acetate buffer (pH 3.6) in a volume ratio of 1 : 1 : 10, respectively. Absorbance after a 4 min incubation was measured at λ = 593 nm by spectrophotometry (Ultrospec 2100 pro; GE Healthcare Life Sciences, Little Chalfont, UK; [57 (link)]). Ferrous sulphate (FeSO4, in water) was used as a reference standard.
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8

Quantification of Sodium-Dependent Phosphate Transporters

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Total RNA was extracted using TRNzol-A + (TIANGEN, Beijing, China). The concentration of total RNA was estimated by a spectrophotometer (Ultrospec 2100 pro, GE Healthcare, Chicago, IL, USA), and the purity was determined by agarose gel electrophoresis. Five hundred nanagrams of total RNA were reverse transcribed into cDNA using the Fast Quant RT Kit (with gDNase) (TIANGEN). qPCR was conducted using the iCycler iQ5 system. The specific primers for NaP-IIb, type III sodium-dependent phosphate cotransporter-1,2 (PiT-1, 2) and β-actin are listed in Table 2. β-actin was used as internal reference gene. Relative gene expression was calculated using the 2-ΔΔCt method [24 (link)]. All the samples were analysed in triplicate, and the operational program for qPCR strictly followed the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) [25 (link)].
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9

Algae Total RNA Extraction

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Total RNA was extracted from 1 g frozen algae using 10 ml of the phenol-based TRI Reagent (Sigma-Aldrich, St. Louis, MO) and 3 ml trichloromethane. RNA was precipitated from the aqueous phase by addition of isopropanol. All preparation and handling steps of RNA took place under RNase-free conditions. After final washing steps with ethanol, RNA was dissolved in RNase-free water and stored at -70°C until used. RNA quantity and purity were determined by measuring the 260/280 ratio using an UV/visible spectrophotometer (Ultrospec 2100 pro, GE Healthcare, Uppsala, Sweden). The quality of RNA was verified by electrophoresis of 0.5 μg RNA in a 1.3% agarose-formaldehyde gel.
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10

Spectrophotometric and Fluorometric NEM Quantification

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NEM concentration in solution was measured by spectrophotometry at 300 nm using an Ultrospec 2100 pro (GE Healthcare, Little Chalfont, UK). The limit of detection by spectrophotometry in our buffer system was determined to be ˜40 μM. To calculate NEM concentrations in the range of 0.1-100 μM a fluorometric maleimide quantification kit (ab112141, Abcam, Cambridge, UK) was used in conjunction with a Varioskan LUX plate reader (ThermoFisher).
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