HPLC analysis was employed to determine the composition of EEAI. The eluted fractions were characterized based on their retention time in comparison to the reference standard ovatodiolide (ARJIL Pharmaceuticals LLC Ltd., Hsinchu, Taiwan), anisomelic acid (ARJIL Pharmaceuticals LLC Ltd., Hsinchu, Taiwan), and apigenin (Chengdu Must Bio-Technology Co., Ltd., Chendu, China). The constituents were characterized by utilizing a photodiode array detector and comparing them with standard UV spectra at a wavelength of 220 nm. A TSK gel Tosoh ODS-80Tm column (250 × 4.6 mm i.d., 5 µm) (Tosoh, Yamaguchi, Japan) in reversed phase was employed for compound separation. During the interval of 0–8 min, a mixture of acetonitrile (J.T Baker, Phillipsburg, NJ, USA) and 0.1% acetic acid (Cascina Favaglie, Milan, Italy) in water (64:36) was used as the mobile phase while keeping the flow rate consistent at 1 mL/min [49 (link)].
Tskgel ods 80tm column
Manufactured by Tosoh
Sourced in Japan
The TSKgel ODS-80TM column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with C18 functional groups, providing a reliable and reproducible platform for reversed-phase chromatography.
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Lab products found in correlation
C17 s1p, by Avanti Polar Lipids (2 mentions)
Acetonitrile, by Avantor (1 mentions)
Apigenin, by Chengdu Must Bio-Technology (1 mentions)
High performance liquid chromatography (hplc), by Hitachi (1 mentions)
Fty720 phosphate, by Cayman Chemical (1 mentions)
Fty720 s phosphate, by Cayman Chemical (1 mentions)
Lc 20ad hplc system, by Shimadzu (1 mentions)
Mightysil rp 18 column, by Kanto Chemical (1 mentions)
14 protocols using tskgel ods 80tm column
1
HPLC Analysis of EEAI Composition
2
HPLC Analysis of Compound Separations
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The composition of SC was analyzed using HPLC (Hitachi Ltd., Tokyo, Japan). The eluted fractions were determined as a function of the retention time compared to the reference standard used. The identification of the components was also confirmed using a photodiode array detector by comparison to standard UV spectra at a wavelength of 254 nm. Compound separations were achieved on a 250 × 4.6 mm i.d., 5 µm reversed-phase TSKgel Tosoh ODS-80Tm column (Tosoh, Yamaguchi, Japan) with acetonitrile (A) and 0.4% acetic acid (B) as the mobile phases, according to the following linear gradient: 0–20 min, 2–10% A, 98–90% B; 20–30 min, 10–25% A, 90–75% B; 30–35 min, 25–2% A, 75–98% B; 35–40 min, 2–2% A, 98–98% B. The flow rate was 1 mL/min.
3
In Vitro GEM Release from PLLA Sheets
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Approximately 10 mg of 10 wt% GEM-containing PLLA non-woven sheet was cut into rectangles and immersed in phosphate-buffered saline (PBS) in a sample tube. The sample tube was placed on a bio shaker and shaken at 100 rpm at 37 °C. At a specific time point (0 h, 1 h, 4 h, 24 h, 7 d, 30 d, or 60 d), the non-woven sheet was removed and transferred to a new sample tube. Chloroform (1 mL) was added to the sample tube to dissolve the sheet, and then 1 mL of water was added to it. After proper stirring, the aqueous layer was drained off and high-performance liquid chromatography (HPLC) measurements were performed. The separation was performed on an TSKgel ODS-80Tm column (Tosoh, Osaka, Japan) using 0.1 M NaCl solution. The solution was passed through a 0.45-μm filter before being transferred to the sample tube. A GEM peak was observed at approximately 14 min of elution. The GEM concentration was calculated based on the peak area. To determine the quantity of GEM released, the measured GEM quantity was subtracted from the initial quantity.
4
Dopamine Quantification in Differentiated Neurons
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Dopamine was analyzed using HPLC as previously reported [18 (link)]: HAP stem cells that differentiated to dopaminergic neurons were lysed with PCA buffer (Perchloric acid 0.2 M, EDTA-2Na 100 μM), then measured by HPLC, using a TSK gel ODS-80TM column (TOSOH BIOSCIENCE, Tokyo, Japan) and an electrochemical detector system EDC-100 (EICOM, Kyoto, Japan).
5
Oligosaccharide Binding Assay of Lectins
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The oligosaccharide binding activity of rKAAs was determined by a centrifugal ultrafiltration HPLC method as described in Hori and Hirayama (2014 ). Briefly, 90 μl of 500 nM His-rKAA-1 or rKAA-1 in 50 mM Tris–HCl (pH7.0) and 10 μl of 300 nM PA-oligosaccharide were mixed and kept at room temperature for 60 min. Subsequently, unbound PA-oligosaccharides were collected by centrifugation (10,000g, 30 s) using Nonosep 10K Device (Pall, NY, USA). An aliquot of the filtrate was applied to a TSKgel ODS 80TM column (4.6 × 150 mm, Tosoh) and unbound PA-oligosaccharide was quantified from the peak area. The amount of bound PA-oligosaccharide was obtained by subtracting the amount of unbound oligosaccharide from the amount of added oligosaccharide, which was determined from the filtrate of the reaction solution without a lectin. The binding activity (%) was calculated as the ratio of the amount of bound oligosaccharide to that of added oligosaccharide.
6
Quantification of S1P and C17S1P in Plasma, Medium, and Liver
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The contents of S1P and C17S1P in the plasma, medium, and liver were determined using two-step lipid extraction followed by HPLC separation, as described previously [21 (link)]. Briefly, samples were sonicated in 3 mL of methanol/chloroform (2:1) with an internal standard for 30 minutes. After adding 2 mL of chloroform, 2.1 mL of 1 mM KCl, and 100 μL of 3 N NaOH, the samples were centrifuged and the alkaline upper phase (3.8 mL) was collected into new tubes, to which 4 mL of chloroform and 200 μL of concentrated HCl were then added. The resulting lower chloroform phases (3.5 mL) formed under these new acidic conditions were collected and evaporated under nitrogen gas and resolved in methanol, followed by HPLC separation using a TSKgel ODS-80TM column (0017202; Tosoh, Tokyo, Japan). For the measurement of the S1P content, we used C17-S1P (860641P; Avanti Polar Lipids) as an internal standard, while for the C17-S1P content, we used FTY720-phosphate (10006408; Cayman Chemical, Ann Arbor, MI).
7
Deglucosylation of Glycans by Glucosidases
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The deglucosylation of PA-G3M9A’ (PA-Glc3Man9GlcNAc1) and PA-G2M9A’ (PA-Glc2Man9GlcNAc1) by the Gls1-only and/or Gls2-only microsomes was checked by size-fractionation HPLC as reported previously [13 (link)]. PA-labeled FNGs from the glucosidase mutant cells were separated by size-fractionation HPLC with a Shodex NH2P-50 4E column (4.6 × 250 mm; Shodex), as reported previously [15 (link)]. To further separate the isomers of the PA-glycans, each PA-glycan fraction, which had been separated by the size fractionation HPLC, was re-injected in reversed-phase HPLC using a TSK-gel ODS-80TM column (4.6 × 150 mm; TOSOH, Tokyo, Japan) as described previously [15 (link)]. FNGs were quantitated from the HPLC profile using standard PA-glucose hexamer (PA-Glc6; 2 pmol/μl) in the standard PA-glucose oligomer (degree of polymerization = 3–15; Takara) as a quantitation reference.
8
Quantification of S1P and dhS1P
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The contents of S1P and dihydrosphingosine-1-phosphate (dhS1P) in the plasma, liver and kidneys were determined using two-step lipid extraction followed by high-performance liquid chromatography separation, as described previously16 (link). Briefly, samples were sonicated in 3 mL of methanol/chloroform (2:1) with an internal standard for 30 min. After adding 2 mL of chloroform, 2.1 mL of 1 mmol/L KCl, and 100 μL of 3 N NaOH, the samples were centrifuged and the alkaline upper phase (3.8 mL) was collected into new tubes, to which 4 mL of chloroform and 200 μL of concentrated HCl were added. The resultant lower chloroform phases (3.5 mL) that were formed under these new acidic conditions were collected and evaporated under nitrogen gas, and resolved in methanol, followed by high-performance liquid chromatography separation using a TSKgel ODS-80TM column (Tosoh, Tokyo, Japan). For the measurement of the S1P contents, we used C17-S1P (Avanti Polar Lipids) as an internal standard, whereas for the C17-S1P contents, we used FTY720(S)-phosphate (Cayman Chemical, Ann Arbor, MI, USA).
9
Oligosaccharide Binding Assay for rKAAs
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The oligosaccharide binding activity of rKAAs was determined by a centrifugal ultrafiltration-HPLC method as described in Hori and Hirayama (2014 ). Briefly, 90 μl of 500 nM His-rKAA-1 or rKAA-1 in 50 mM Tris–HCl (pH7.0), and 10 μl of 300 nM PA-oligosaccharide were mixed and kept at room temperature for 60 min. Subsequently, unbound PA-oligosaccharides were collected by centrifugation (10,000g, 30 s) using Nonosep 10 K Device (Pall, NY, USA). An aliquot of the filtrate was applied to a TSKgel ODS 80TM column (4.6 × 150 mm, Tosoh), and unbound PA-oligosaccharide was quantified from the peak area. The amount of bound PA-oligosaccharide was obtained by subtracting the amount of unbound oligosaccharide from the amount of added oligosaccharide, which was determined from the filtrate of the reaction solution without a lectin. The binding activity (%) was calculated as the ratio of the amount of bound oligosaccharide to that of added oligosaccharide.
10
Isolation of Aspergillus Oryzae Metabolites
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Berkeleyone A (3 ), preaustinoid A2 (6 ), and preaustinoid A3 (7 ) were isolated from the culture of the Aspergillus oryzae NSAR1 (niaD−, sC−, ΔargB, and adeA−) transformants expressing AusB, AusE, and AusC. A. oryzae NSAR1 transformants co-expressing PrhI and PrhJ was used to prepare preaustinoid A1 (5 ). The culture medium was extracted with ethyl acetate, concentrated, and purified by silica-gel chromatography (Wakogel C-200, 0 to 5% methanol in chloroform). The fraction containing the target compound was concentrated and purified by Shimadzu Prominence HPLC system using TSK-gel ODS-80TM column (Tosoh Co. Ltd., 7.8 i.d. × 300 mm, 50% acetonitrile, 3.0 ml min−1)16 (link), 18 (link).
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