Upon reaching 80–90% confluence, ADSCs at passage 3 were rinsed with PBS and cultured for 48 h in serum-free endothelial cell growth medium (EGM)-2 MV (Lonza, Walkersville, MD, USA). The conditioned medium was collected and centrifuged to separate EVs, following the previously constructed protocols (20 (link), 21 (link)). In brief, the conditioned culture medium was centrifuged at 300 g for 10 min, at 2,000 g for 10 min, and then at 10,000 g for 30 min to remove the remaining cells and cell fragments by using a 0.22-μm filter (Millipore, Billerica, MA, USA). After that, EVs (Optima L-90K ultracentrifuge; Beckman Coulter, Brea, CA, USA) were purified by 2-h ultracentrifugation at 100,000 g at 4°C. Finally, the EVs were separated by particle size fractionation and concentrated by centrifugation using a centrifuge tube (Millipore) with a molecular weight cut-off value of 100 kDa. The obtained EV particles were resuspended in PBS and stored at -80°C. Nanoparticle tracking analysis (NTA, Zeta View PMX 110, Particle Metrix, Meerbusch, Germany) and transmission electron microscope (TEM, JEOL microscope, JSM-7001TA, Tokyo, Japan) were implemented to analyze the diameter and ultrastructure of EVs. Western blot assay was performed to determine the surface protein markers CD9, CD63, and CD81 to identify EVs.
Centrifuge tube
Manufactured by Merck Group
Sourced in United States
A centrifuge tube is a type of laboratory equipment used to separate different components within a liquid mixture through centrifugal force. It is a cylindrical container designed to be placed in a centrifuge, a device that spins the tube rapidly to create this force and facilitate the separation process.
Automatically generated - may contain errors
Lab products found in correlation
Zetaview pmx 110, by Particle Metrix (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Optima l 90k ultracentrifuge, by Beckman Coulter (1 mentions)
Egm 2mv, by Lonza (1 mentions)
Dialysis bag, by Yuanye Bio-Technology (1 mentions)
Anhydrous ether, by Sinopharm (1 mentions)
Cholesterol, by Maclin Biochemical Technology (1 mentions)
2 hydroxy 4 2 hydroxyethoxy 2 methyl propiophenone, by J&K Scientific (1 mentions)
Lecithin, by Maclin Biochemical Technology (1 mentions)
Gemcitabine hydrochloride, by Meilun (1 mentions)
Ammonium persulphate, by Merck Group (1 mentions)
Chaps, by Merck Group (1 mentions)
Bis acrylamide, by Merck Group (1 mentions)
Temed, by Merck Group (1 mentions)
Tris base, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
6 protocols using centrifuge tube
1
Extracellular Vesicle Isolation from ADSC Secretome
2
Liposomal Gemcitabine Delivery System
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The following materials are used in the study: Lecithin (CAS: 8002-43-5 from Shanghai Macklin Biochemical Co., Ltd); Cholesterol (CAS:57-88-5 from Shanghai Macklin Biochemical Co., Ltd); Gemcitabine hydrochloride (CAS:122111-03-9 from MeilunBio); Anhydrous ether (CAS:20161103 from Sinopharm Chemical Reagent Co., Ltd); 0.22 μm, 0.45 μm sterile syringe filter (from i-Quip); Gelatin (CAS:180LB8 from Rousselot Gelatin Co. Ltd); Methacrylic anhydride (CAS:760-93-0 from Aladdin); Dialysis bag (Mw: 8000–14 000, 3500 from Shanghai Yuanye Bio-Technology Co., Ltd); Centrifuge tube (Mw: 3000 from Millipore); and 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone (CAS:106797-53-9 from J&K Scientific).
3
Yogurt Syneresis Measurement Protocol
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ISIRI 2582 standard [23] was used as the criteria to measure pH and ISIRI 695 standard [24] was used to test the acidity of samples. To measure the syneresis, a process taken from [4] , with some modifications, was followed. Nevertheless, the syneresis was reported as g of the whey isolated from the entire weight (=100 g) of yogurt. The 25-g yogurt sample of each batch was weighed with a centrifuge tube (Sigma, USA). It was then centrifuged at 3500× g at 4 °C for 10 minutes. The supernatant was disposed of the whey extracted from the sample and the resultant yogurt in the centrifuge tube was then weighed again. The rate of the drained yogurt weight to the entire weight (100 g) of yogurt prior to the centrifuge was defined as syneresis.
4
Comprehensive Protein Extraction and Purification
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TCA (Trichloro Acetic acid, Sigma-Aldrich, USA), Acetone(Mecrk, India), Urea (Sigma-Aldrich, USA), Thio Urea( Sigma-Aldrich, USA), CHAPS(Sigma -Aldrich, USA), ASB-14 (Merck, India), Dithithretol( Sigma-Aldrich, USA), Bromophenol blue (Merck, India), H2O (HPLC purified ,Merck, India), Acrylamide( Sigma -Aldrich, USA), Bis-Acrylamide(Sigma-Aldrich, USA), Tris base( SRL,India), Hydrochloric acid(Merck, India), Sodium dodecyl sulphate( Merck, India), Ammonium per sulphate( Sigma-Aldrich, USA),TEMED( Sigma-Aldrich, USA),Glycerol( Merck,India), β-Mercaptoethanol( Sigma-Aldrich, USA), Coomassie brilliant blue-R250( Merck, India), Acetic acid(Merck, India), Methanol(Merck,India),-20 0 C Refrigerator, Centrifuge tube(Sigma G 3-18K), Chloroform( Merck,India), Methanol(Merck, India, Isopropanol (Merck, India), Polyethylene Glycol-6000(Merck, India).
5
Purification and Characterization of Recombinant GOT1
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The expression construct pET26b-got1 (Invitrogen, Carlsbad, NJ, USA) was C-terminal His6-tagged and transformed into E. coli BL21 (DE3) cells at 30 °C for 6 h. The recombinant protein was initially passed over a HisTrap™ FF crude column (GE Healthcare, 5 mL, Pittsburgh, PA, USA). Peak elution fractions containing GOT1 were collected and concentrated using centrifuge tubes (Millipore, Billerica, MA, USA) with a 10 kDa cutoff, and then purified via anion-exchange chromatography with Resource Q (GE Healthcare, 5 mL, Pittsburgh, PA, USA). Subsequently, the sample was injected onto a Superdex200 Increase 10/300 GL Column (GE Healthcare, Pittsburgh, PA, USA) and dialyzed in 20 mM HEPES buffer (pH 7.5) and 200 mM NaCl.
6
Albumin-Based Nanoparticle Formulations
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NPs were formed according to protocols described below. Briefly, 120 mg of HSA was added to 60 mL of deionized water and stirred for 10 min. Then, IR780, which was dissolved in ethanol, was slowly added to the HSA solution to form NPs. To remove free IR780, the NPs were hyper-filtrated by a 30 kDa Millipore filter. Centrifugation (1438 × g, 5 min) was used to remove large aggregates of IR780 post ultrafiltration.
To prepare ONPs and PNPs, an unfolding/self-assembling method was adopted36 . Briefly, 100 mg of HSA was mixed with 3 mL of deionized water and stirred for 5 min. Specified quantities of IR780, PFTBA or oil were added to the HSA solution. ONPs and PNPs were formed under sonication at 300 W in an ice bath for 10 min. Free IR780 was removed by ultrafiltration in centrifuge tubes (Millipore) for 10 min.
To prepare ONPs and PNPs, an unfolding/self-assembling method was adopted36 . Briefly, 100 mg of HSA was mixed with 3 mL of deionized water and stirred for 5 min. Specified quantities of IR780, PFTBA or oil were added to the HSA solution. ONPs and PNPs were formed under sonication at 300 W in an ice bath for 10 min. Free IR780 was removed by ultrafiltration in centrifuge tubes (Millipore) for 10 min.
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