The PCR primers introduce the full-length adaptor sequence required for Illumina sequencing (for details see Illumina small RNA PCR primers). PCR was performed in 12.5μl using half of the ranhexRT sample as a template [1X KAPA HiFi HotStart ReadyMix (KapaBiosystems #KK2602), 400 nM each primer]. The final PCR amplified library was submitted to two consecutive 1x NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel #7449970.5) according to manufacturer’s instructions. The final library was eluted in 20μl of 10 mM Trizma hydrochloride solution (Sigma-Aldrich #T2319–1L).
Trizma hydrochloride solution
Manufactured by Merck Group
Sourced in United States
Trizma hydrochloride solution is a buffering agent used in various laboratory applications. It is a water-soluble, inorganic compound that helps maintain a stable pH environment in chemical reactions and biological assays.
Automatically generated - may contain errors
Lab products found in correlation
Ah111501, by GE Healthcare (2 mentions)
1 13c pyruvic acid, by Merck Group (2 mentions)
Nucleomag ngs clean up and size select magnetic beads, by Macherey-Nagel (1 mentions)
Small rna pcr primers, by Illumina (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
Magnesium chloride solution, by Merck Group (1 mentions)
Bovine serum albumin solution, by Merck Group (1 mentions)
Kinase glo luminescent kinase assay, by Promega (1 mentions)
N 1 paper, by Cytiva (1 mentions)
Spinlab polarizer, by GE Healthcare (1 mentions)
10 n sodium hydroxide solution, by Thermo Fisher Scientific (1 mentions)
Ethylenediaminetetraacetic acid edta, by Merck Group (1 mentions)
Polyvinyl alcohol (pva), by Merck Group (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Span 80, by Merck Group (1 mentions)
6 protocols using trizma hydrochloride solution
1
Illumina Sequencing Library Preparation
2
ROCK Kinase Inhibition Assay
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The ROCK kinase assay is a luminescent kinase assay that measures IOP formed from a kinase reaction. ADP is converted into ATP and is converted into light by Kinase-Glo Luciferase. The Kinase-Glo Assay can be used to monitor the activity of virtually any ADP-generating enzyme. The relative light unit (RLU) without test compound was set as 100% (blank value), and that without enzyme and compound was set as 0% (normal value). The reaction rate (% of black) was then calculated from the RLU.
The following reagents were used in this assay: ROCK1 (750 ug/mL, Carna Biosciences, Cat.01-109); long S6 kinase substrate peptide (Millipore, Cat#12-420); magnesium chloride solution (Sigma, 60142-100 mL); EGTA solution (0.5 M pH 8.0, Bioword, Cat. 4052008-1); Trizma® hydrochloride solution (1 M, pH 7.5 Sigma, T2319-100 mL); bovine serum albumin solution (20 mg/mL in water, Sigma); Kinase-Glo luminescent kinase assay (Promega, RV6712); ripasudil (K115) hydrochloride dehydrate (Selleckchem, S7995-5 mg); ATP solution (100 mM, GE Healthcare, Cat.27-2056-010). A ROCK kinase inhibitor k-115 was used as the positive control.
Max = observed enzyme activity measured in the presence of enzyme, substrate(s), and cofactors utilized in the method.
Min = Normal value in the method
The following reagents were used in this assay: ROCK1 (750 ug/mL, Carna Biosciences, Cat.01-109); long S6 kinase substrate peptide (Millipore, Cat#12-420); magnesium chloride solution (Sigma, 60142-100 mL); EGTA solution (0.5 M pH 8.0, Bioword, Cat. 4052008-1); Trizma® hydrochloride solution (1 M, pH 7.5 Sigma, T2319-100 mL); bovine serum albumin solution (20 mg/mL in water, Sigma); Kinase-Glo luminescent kinase assay (Promega, RV6712); ripasudil (K115) hydrochloride dehydrate (Selleckchem, S7995-5 mg); ATP solution (100 mM, GE Healthcare, Cat.27-2056-010). A ROCK kinase inhibitor k-115 was used as the positive control.
Max = observed enzyme activity measured in the presence of enzyme, substrate(s), and cofactors utilized in the method.
Min = Normal value in the method
3
Water Solubility Determination of Films
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The tested films were cut into strips about 2 × 4 cm and immersed in 20 mL of a pH 7.0 aqueous solution containing 10% (w/w) 1M Trizma® hydrochloride solution pH 7.0 (Sigma-Aldrich). The immersed film was kept at room temperature for 24 h. Afterward, the film fragments were recovered by filtration using Whatman N°1 paper and dried in an oven at 105 °C for 24 h. At the same time, a set of identical film sheets was dried under the same conditions without first dissolving.
Water solubility (WS) was calculated as:
where m1 is the mass in grams of the dry films, and m2 is the mass in grams of the solubilized films.
Water solubility (WS) was calculated as:
where m1 is the mass in grams of the dry films, and m2 is the mass in grams of the solubilized films.
4
Polarized [1-13C]Pyruvate Preparation
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We used a SPINlab polarizer (GE) to polarize [1-13C]pyruvate. The preparation steps for polarization are as follows: (i) The 13C-enriched pyruvate sample was prepared with a stable organic radical: 15 mM AH-111501 (GE) was mixed in [1-13C]pyruvic acid (Sigma-Aldrich, 677175) thoroughly. (ii) Buffer solution for dissolution was prepared: 0.4 mM EDTA was added in 40 mM Trizma hydrochloride solution (Sigma-Aldrich, T2663). (iii) The pyruvate sample (100 μl) from step (i) and 20 ml of the buffer solution from step (ii) were loaded into the SPINlab polarizer (3.35 T, 0.98 K). After 90 min of polarization, the pyruvate sample was quickly dissolved into an ice-cold flask with 120 μl of 10 N sodium hydroxide solution (Fisher Scientific, SS255) to make the dissolved sample neutral (pH ~ 7.4) and reach 37°C faster. The dissolved pyruvate sample was added into the cell suspension with a ratio of 1:10, which made the concentration of pyruvate 14 mM.
5
Hyperpolarized [1-13C] Pyruvate Sample Prep
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We used the dDNP method to hyperpolarize [1-13C] pyruvate. The following describes the sample preparation steps: (1) [1-13C] pyruvic acid (Sigma-Aldrich, St. Louis, MO, USA) was thoroughly mixed with 15 mM AH-111501 (GE Healthcare, Chicago, IL, USA), 10 μL of which was loaded into the SPINlab polarizer (5 T, 0.87 K, GE Healthcare, Chicago, IL, USA). (2) 100 mM Trizma hydrochloride solution (Sigma-Aldrich, St. Louis, MO, USA) with 1 mM ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, St. Louis, MO, USA) was prepared in deuterium oxide (D2O) as dissolution buffer solution [24 (link)]. (3) After 120-min polarization, the pyruvate sample was quickly dissolved into a flask and neutralized with 13 μL of 10 N sodium hydroxide solution (Fisher Scientific, Waltham, MA, USA). The sample was then mixed with cell suspension, which was prepared right before the dissolution, with a ratio of 1 to 4 (10 μL pyruvate sample and 40 μL cell suspension); this made the final concentration of [1-13C] pyruvate approximately 5 mM.
6
Fabrication of PLGA Microcarriers with PDA Coating
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For the fabrication of the PLGA microcarrier, PLGA (with a molecular weight of about 66,000–107,000), dichloromethane (DCM), Span®® 80, PVA (with a molecular weight of ~61,000), gelatin (from porcine skin; gel strength 300; Type A), dopamine hydrochloride, Trizma®® hydrochloride solution (pH 8.0; BioPerformance Certified; 1 M; suitable for cell culture; from Sigma-Aldrich), and iron(II,III) oxide (nanopowder; with a particle size of 50–100 nm (SEM); 97% trace metal basis) were purchased from Sigma-Aldrich (St. Louis, MO, USA). For the PDA coating on the microscaffolds, dopamine hydrochloride was purchased from Sigma-Aldrich.
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