The aptamer selected for the CCRF-CEM cells, sgc8c, (5′-FTIC-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA-NH2-3) and library DNA containing a randomized sequence of 41 nucleotides were synthesized at a μmol scale using an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). Both sgc8c and DNA library were coupled with fluorescein isothiocyanate dye (FTIC) modifier at the 5’ end and amine (-NH2) modifier at the 3’ prime end during synthesis. After synthesis, they were deprotected in AMA (ammonium hydroxide /40% aqueous methylamine, 1:1) at 65°C for 30 min. After deprotection, all sequences were purified by reversed-phase HPLC (ProStar-Varian, Walnut Creek, CA) with a C18 column (Econosil, 5μm, 250mm length, 4.6mm diameter) from Alltech (Deerfield, IL) using a mobile phase containing 100 mM triethylamine acetic acid buffer (TEAA, pH 7.5) and acetonitrile (0-30min, 10-00%). All purified DNA solutions were dried in acid-resistant centriVap centrifugal vacuum concentrators (Labconco, Kansas City, MO). The dried DNA at the bottom of 2 mL tubes was dissolved in 50-100μL of DNA grade water. The concentrations of all DNA were determined by measuring the absorbance values at 260 nm.
Centrivap centrifugal vacuum concentrator
Manufactured by Labconco
Sourced in United States
The CentriVap Centrifugal Vacuum Concentrator is a laboratory instrument designed to gently remove solvents or water from liquid samples through the process of centrifugation and vacuum evaporation. It provides a controlled environment to concentrate and preserve samples for further analysis or processing.
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9 protocols using centrivap centrifugal vacuum concentrator
1
CCRF-CEM Cell Aptamer Synthesis
2
NMR-based Metabolomics Analysis of Rice
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For each of the three groups (i.e., WT, M, and ECE), 10 biological replicates of rice plants were used for the extraction and subsequent NMR-based metabolomics analysis. Each rice plant was extracted individually using a method modified from previous reports [23 (link),54 (link)]. Firstly, the rice sample-containing tube was snap-frozen in liquid nitrogen and the rice tissue was then swiftly ground into fine powder using a pre-cooled pestle. Pre-cooled methanol/water (v/v = 2/1, −20 °C) was added into the homogenized sample at a ratio of 600 µL per 100 mg powder. Afterwards, the mixture was further homogenized using a 2010 Geno/Grinder® (SPEX Sample Prep, Metuchen, NJ, USA) at 1300 rpm for 90 s. Then the homogenate mixture was sonicated in an ice bath with 10 cycles of 30 s sonication and 30 s break. Following centrifugation (14,489× g, 10 min, 4 °C), the supernatant was collected, and the remaining pellets were further treated twice using the same procedure. Three supernatants were combined and lyophilized after removal of methanol in vacuo (CentriVap Centrifugal Vacuum Concentrators, Labconco, MO, USA). Each dried extract was reconstituted into 600 μL phosphate buffer prepared as previously described. After a final centrifugation, 500 μL supernatant was transferred into a 5 mm NMR tube for NMR analysis.
3
Cell Metabolite Extraction and Storage
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Cell sample preparation. Cell culture plates were washed with PBS and snap-frozen in liquid nitrogen and stored at -80˚C. To the culture plate, 1 ml of -20˚C pre-cooled 80% methanol [with internal standard mixture of carnitine C2:0-d3, carnitine C10:0-d3, carnitine C16:0-d3, LPC 19:0, free fatty acid (FFA) 16:0-d3, FFA 18:0-d3, chenodeoxycholic acid-d4, cholic acid-d4, leu-d3, phe-d5 and tryptophan-d5] was added, and cells were gently scraped off the bottom of the plate into a 5-ml EP tube using a cell scraper. After thorough vortexing and centrifugation (13,000 x g for 10 min at 4˚C), the supernatant was pipetted for drying into a CentriVap Centrifugal Vacuum Concentrator (Labconco Corp., Kansas City, MO, USA). The dried residues were stored at -80˚C until analysis.
4
Serum Metabolite Extraction Protocol
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Fifty microliters of serum was mixed with 200 µL ice-cold methanol containing IS in a 1.5 mL Eppendorf tube. The mixture was vortexed for 30 s for protein precipitation and metabolite extraction, placed on ice for 10 min, then centrifuged at 14,000 ×g and 4 °C for 15 min. A total of 180 µL of supernatant was collected then transferred to a new 1.5 mL Eppendorf tube for vacuum concentration at 4 °C using a CentriVap Centrifugal Vacuum Concentrator (Labconco, MO, USA). The dried residue was reconstituted in 80 µL water/acetonitrile 4:1 (v/v) and the solution was further centrifuged at 14,000 ×g and 4 °C for 15 min, and the supernatant was used for liquid chromatography-mass spectrometry (LC-MS) metabolite analysis.
5
Metabolic Profiling of ASCT2-Depleted Cells
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HCT116 cells were infected with a control or ASCT2 shRNA and selected with puromycin for 48 h, and then switched to fresh medium for another 24 h. Cells were rinsed with 1 ml ice-cold PBS and quenched with 1 ml 80% methanol (methanol: water=4:1, v/v). Cells were collected in tubes by scraping with a pipette and the extracts were vortexed for 5 min. Samples were centrifuged at 14 000 g for 15 min, and the supernatant was transferred to a new tube for evaporation in a CentriVap Centrifugal Vacuum Concentrator (Labconco, Kansa, MO, USA). The dried metabolites were dissolved in 50 μl methoxyamine pyridine (20 mg/ml), vortexed for 30 s, and ultrasound-treated for 10 min. Oximation was then conducted at 40 °C for 2 hr, followed by silylation with 40 μl MSTFA for 1 hr. After derivatization, the solution was centrifuged at 14 000 g for 15 min and diverted to a 2 ml glass vial. Metabolic profiling was performed by GCMS-QP 2010 analytical system (Shimadzu, Tokyo, Japan) as previously described.39 (link)
6
Quantification of Plasma and Tissue TN
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The concentration of TN in plasma and tissue was analyzed by high performance liquid chromatography (HPLC) using a P230 pump and a UV230 UV/Vis detector (Dalian Elite Analytical Instruments Co., Ltd., Liaoning, China) and separated using a Hypersil® BDS C18 column (200 mm × 4.6 mm) containing particles measuring 5 µm in diameter at 30 °C. The ultraviolet wavelength was 264 nm. The mobile phase was methanol/isopropanol (80/20, v/v) at a flow rate of 1 ml/min. Before the analysis, the plasma samples and tissue samples were treated as follows: 100 µl of the plasma samples or homogenates (equivalent to 0.1 g tissue) was mixed with methanol (100 µl), internal standard (100 µg/ml VE) (100 µl) and n-hexane (600 µl). The entire mixture was vortexed for 5 min and centrifuged at 10,000 rpm for 10 min. The supernatant (500 µl) was dried using a CentriVap® Centrifugal Vacuum Concentrator (Labconco Corporation, USA) and dissolved in the mobile phase (100 µl). The resulting mixture was vortexed for 1 min and centrifuged at 10,000 rpm for 10 min. The supernatant (20 µl) was collected and used for the HPLC analysis.
7
Quantifying Gibberellic Acid in Leaf Samples
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The sample leaves were harvested and stored at −80°C. The GA extraction was followed as the description with some modification (Baba et al., 2019 ). Approximately 100 mg leaves were ground by liquid N2 and incubated with 1 ml of 80% methanol solution containing 0.01% butylhydroxytoluene overnight at 4°C. After centrifugation at 11 758 g (Eppendorf centrifuge 5415D; Marshall Scientific, Hampton, NH, USA) for 10 min at 4°C, the supernatant was removed and mixed with hexane. The bottom layer was collected and dried by CentriVap Centrifugal Vacuum Concentrator (Labconco Corp., Kansas City, MO, USA). The dried pellet was resolved in 200 µl PBS for detection. To determine the concentration of GA, we followed the protocol of the enzyme‐linked immunosorbent assay kit (CEA759Ge; ELISA Kit for Gibberellic Acid (GA); Cloud Clone Corp., Katy, TX, USA). Approximately 50 µl of each sample was loaded into one well and incubated at 37°C after the regent was added. After the reaction, the signal was measured at 450 nm immediately by spectraMax M2 (Molecular Devices, San Jose, CA, USA). Three biological repeats were performed for total GA concentration measurement.
8
Quantification of NAD+ Metabolites
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For absolute quantification of the intermediates implicated in NAD+ de novo synthesis and salvage pathway, samples were prepared by spiking 100 µl of tissue extract with 25 µl of internal standard solution and then sample extracts were evaporated to dryness using a centrivap centrifugal vacuum concentrator (Labconco). Dry extracts were subsequently reconstituted with 75 µl of water and injected into the LC‐MS/MS system.
9
In-Gel Tryptic and Chymotryptic Digestion of Proteins
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Three coomassie blue stained SDS-PAGE gel bands corresponding to those targeted by the anti-His-tag antibody in the immunoblots were excised and destained in 25 mM ammonium bicarbonate-50% acetonitrile at least 3 times and dehydrated with acetonitrile, then reduced with 25 mM DTT in 50 mM ammonium bicarbonate at 56 °C for 25 min, and alkylated with 55 mM iodoacetamide at room temperature in the dark for 30 min. After washing and dehydration in 25 mM ammonium bicarbonate-50% acetonitrile and absolute acetonitrile, gel pieces were digested with 10 ng/ μL mass spectrometry grade trypsin gold (Promega, V5280) or chymotrypsin (Promega, V1062) in 25 mM ammonium bicarbonate at 37 °C for 16 hrs. Peptides were extracted sequentially with 20 mM ammonium bicarbonate, 50% acetonitrile and 5% formic acid, and then pooled peptides were evaporated to dryness in acid-resistant CentriVap centrifugal vacuum concentrator (Labconco Kansas City, MO), and re-suspended with 20 μL of 2% methonal-1% formic acid. NanoLC-MSMS analysis of in-gel digested peptide was performed on Waters® nanoACQUITY UPLC® coupled LTQ-Orbitrap Elite ETD Mass Spectrometer (Thermo Fisher Scientific) as previously described [35 (link)].
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