Cell culture supernatants were passed through 0.22 µM sterile filters and were concentrated with Amicon® Ultra-2 mL Centrifugal Filters (MerckMillipore), according to the manufacturer’s instructions. We applied the same starting volume (2 ml) of all culture supernatants to the Amicon® Ultra-2 mL Centrifugal Filters and centrifuged until all samples were reduced to less than 500 µl. Because the procedure reduced all samples to slightly different volumes, we then added varying amounts of fresh reagent diluent to the samples, to adjust all samples to the equal volume of 500 µl. To take this 4-fold concentration into account, we divided the ELISA-readout values by 4 and reported these final values in Figures 2 3
Amicon ultra 2 ml centrifugal filters
Manufactured by Merck Group
Sourced in Germany, United States
The Amicon® Ultra-2 mL Centrifugal Filters are a laboratory filtration device designed to concentrate and purify samples. They utilize a low-binding regenerated cellulose membrane to separate and retain molecules based on their size.
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Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Human mica duoset elisa kit, by R&D Systems (1 mentions)
Elisas, by Cloud-Clone (1 mentions)
Complete ultra mini protease inhibitor, by Roche (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
Enrich q 10 100 column, by Bio-Rad (1 mentions)
Polycarbonate membrane, by Cytiva (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Colloidal coomassie blue stain, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Image studio lite software v5, by LI COR (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Econo pac chromatography column, by Bio-Rad (1 mentions)
Avanti mini extruder, by Avanti Polar Lipids (1 mentions)
Dioleoylphosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
12 protocols using amicon ultra 2 ml centrifugal filters
1
Concentration of Cell Culture Supernatants
2
Quantifying MSC C3a and SDF-1 Expression
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To confirm the expression of C3a and SDF-1 protein in MSC lysates and to determine whether C3a and SDF-1 are released into the MSC culture media (DMEM supplemented with 10% Foetal Bovine Serum (FBS) and 100U/ml penicillin + 0.1mg/ml streptomycin), MSCs were seeded into Nunclon™ 35mm petri dishes, to mimic the experimental set-up utilised for our direct contact islet-MSC co-culture system [6, 7] . After 2 days, MSCs from each petri dish were trypsinised and resuspended in ice-cold PBS supplemented with cOmplete ULTRA mini protease inhibitors (Roche Diagnostics, Burgess Hill, UK), then sonicated. The MSCconditioned media (CM) from each Petri dish was also collected and concentrated x24 using 3,000NMWL Amicon® Ultra 2ml centrifugal filters (Merck Millipore, Middlesex, UK).
Control samples were MSC culture media alone, which was concentrated x24. C3a and SDF-1 were measured in MSC lysates and CM using ELISAs (Cloud-Clone Corp, Houston, USA and R&D Life Sciences, Abingdon, UK).
Control samples were MSC culture media alone, which was concentrated x24. C3a and SDF-1 were measured in MSC lysates and CM using ELISAs (Cloud-Clone Corp, Houston, USA and R&D Life Sciences, Abingdon, UK).
3
Angiogenesis Assay in Chicken Embryos
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Fertilized chicken eggs laid the day before (purchased from Drost Loosdrecht B.V.) were incubated sideways at 37°C and 65% humidity for 3 days before rupturing the air sack located at the blunt side of the egg, as well as opening a small hole on the top to deflate the air-sack and lower the fluid level. After 3 days of incubation, part of the shell was removed and filters containing concentrated CM were placed on top of the CAM. Therefore, CM harvested from chondrogenically differentiated BMSCs was concentrated utilizing the Amicon® Ultra-2 mL Centrifugal Filters (Merck, Kenilworth, NJ, United States) and a sterile filter disks with a diameter of 5 mm were soaked with 5 μL of the concentrated CM and placed on the membrane. As a negative control, concentrated basal medium was utilized and 100 ng FGF2 was used as positive control (Schindelin et al., 2012 (link)). After 3 days of incubation, the CAM was fixed with 4% Formalin/PBS solution and the removed from the egg. Induction of vessel formation was assessed by taking pictures of each filter on the membrane, and the blinded pictures were ranked by three independent observers. A total of 45 images were ranked (45 being the highest and one the lowest rank). The inter-observer correlation was tested and the final rank determined by averaging the ranks per image.
4
Caspase-3 mediated Nt-acetylation
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Clear lysate extracted under native conditions was split into two aliquots. One was left untreated (control) while the other was mixed with recombinant human caspase-3 in a 1:20 w/w ratio. Both samples were kept at 37 °C with gentle mixing. After 1, 6, and 18 h, a fraction of 200 μg protein was taken from the control and caspase-3-treated samples into a clean tube containing an equal volume of 20 mM Hepes, 8 M guanidium-HCl pH 7.5 and incubated at 95 °C for 5 min. For checking in vitro Nt-acetylation, the clear lysate was split into two aliquots. One was dialyzed by 3 cycles of concentration and 10× dilution at 4 °C in ice-cold 20 mM Hepes pH 7.8, 1 mM DTT, 1 mM EDTA, 100 mM NaCl using 3k cut-off Amicon Ultra 2 ml Centrifugal Filters (Merck). Equal amounts of protein from both dialyzed and undialyzed samples were treated with caspase-3 as described above. Aliquots were taken from each sample after 0.5, 6, and 18 h. Nt-acetylation samples were subject only to HYTANE.
5
Size Exclusion Chromatography Purification
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For size exclusion chromatography, the fractions that contained the protein of interest were pooled and concentrated using Amicon® ultra 2 mL centrifugal filters (Merck Millipore, Burlington, MA, USA) with a 10 kDa cut‐off. For equilibration, the column (Superdex 200 10/300 GL from GE Healthcare, Chalfont, UK) was flushed with 30 mL 1× PBS with a flowrate of 0.5 mL·min−1. The sample was injected and fractions were collected. Fractions containing the protein of interest (2 mL) were further concentrated to 200 µL using Amicon® ultra 2 mL centrifugal filters and quantified by densitometric analyses using known amounts of purified BSA. After estimating the concentrations of both FLNAQ and FLNAR by gel densitometry, the samples were subjected to AFM studies.
6
Recombinant MSA/miR-1291 Production
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Production of recombinant MSA/miR-1291 or control MSA was conducted as described (Li et al., 2015 (link); Wang et al., 2015 (link); Ho et al., 2018 (link)). Briefly, RNA expression plasmids were transformed into HST08 E. coli and incubated in ampicillin-containing (100 μg/ml) LB medium at 37°C overnight. Total bacterial RNAs were extracted using Tris-HCl saturated phenol method and then were analyzed by denaturing urea polyacrylamide gel electrophoresis (PAGE) analysis to verify the expression of recombinant RNAs. Purification of MSA/miR-1291 from total RNA was conducted on an NGC QUEST 10 PLUS fast protein liquid chromatography system (Bio-Rad) consisting of a fraction collector by using an anion exchange Enrich-Q 10 × 100 column (Bio-Rad). Fractions containing pure MSA/miR-1291 or MSA, which were validated by denaturing urea PAGE, were pooled and precipitated by ethanol and then desalted and concentrated by Amicon ultra–2 ml centrifugal filters (30 kDa; EMD Millipore, Billerica, MA). Purity of isolated RNA was quantitatively determined by a high-performance liquid chromatography method (Wang et al., 2015 (link)). RNAs showing high level of homogeneity (> 97%) were used in this study.
7
Preparation and Characterization of DMU-214-Loaded Liposomes
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DMU-214-loaded liposomes were prepared by a thin lipid film hydration method followed by extrusion as described here (Skupin-Mrugalska et al., 2021 (link)) by using chloroform solutions of DMPC, DPPC, POPC (50 mg/ml), POPG (50 mg/ml), DMU-214 (10 mg/ml). DMU-214 was loaded passively into vesicles during hydration. Appropriate volumes of stock solutions were mixed, and then chloroform was evaporated under gradually reduced pressure at 40 °C in a round-bottomed flask. The resulting lipid film was then hydrated in PBS buffer pH 7.4. The resulting liposome suspension was passed 21 times through the polycarbonate membrane (Whatman, Kent, UK) with pore diameters of 100 nm using a syringe extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA). Unbound material was separated from liposomes by fast ultrafiltration using Amicon Ultra 2 ml centrifugal filters with molecular weight cutoff (MWCO) 50 kDa (Merck KGa, Darmstadt, Germany). The amount of DMU-214 incorporated into liposomes was determined by the chromatographic method (paragraph 2.5). Encapsulation efficiency EE (%) was calculated according to Eq. (1): EE (%) = (Cm/Ci) x 100 (1), where Cm is the concentration of DMU-214 loaded into liposomes, determined by HPLC, Ci is the initial (maximum) concentration of DMU-214 added in the liposomal formulation. The experiment was performed in triplicates.
8
Gelatin Zymography for Protease Activity
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The explant culture medium was concentrated (approximately 10-fold) using centrifugal filter units (Amicon Ultra-2 mL Centrifugal Filters, Merck-Millipore, Darmstadt, Germany). Total protein content was quantified (BCA protein assay, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Protein aliquots (10 µg) were separated in 7.5% acrylamide-bisacrylamide gels containing 0.3 mg/mL gelatin from porcine skin (Sigma, St Quentin Fallavier, France) under non-reducing, non-denaturing conditions. Gels were incubated (2 × 30 min) in 2.5% Triton X-100 (Sigma) for protein renaturation and then proteolytic reaction was allowed to take place in 50 mM Tris-HCl, 50 mM NaCl, 10 mM CaCl2, pH 7.5, for 20 h at 37 °C, followed by staining with colloidal Coomassie blue stain (Sigma, St Quentin Fallavier, France). Gelatinolytic activity was visible as clear zones on a blue background. Images were obtained using a Li-Cor Odyssey Infrared Imager (Li-Cor Biosciences, Lincoln, NE, USA) and analyzed with Image Studio Lite Software v5.2 (Li-Cor Biosciences, Lincoln, NE, USA).
9
Extracellular Vesicle Purification with TFF and SEC
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Using TFF methodology, UF samples were concentrated while removing small proteins and other molecules from the sample. Briefly, two syringes, one containing the UF sample were pushed gently up and down to concentrate and purify EV through TFF Easy® filtration unit (HansaBioMed Life Sciences, Tallinn, Estonia). The process was repeated until the sample volume reached to 1 mL, which was then further concentrated up to 500 µL using Amicon® Ultra-2 mL centrifugal filters (10 kDa cut-off, Merck Millipore Ltd, Darmstadt, Germany) by centrifuging at 2000 g.
EV were isolated from the purified and concentrated UF samples using a SEC method (Reshi et al. 2021 (link)). SEC columns were prepared by packing Econo-Pac® Chromatography columns (Bio-Rad, Hercules, CA USA, cat:7321010) with size exclusion chromatography resin (catalog. Sepharose 4 fast flow™, Cytvia, Uppsala, Sweden). In brief, the packed SEC columns were positioned vertically in a holder and washed by running through ultrapure Milli-Q® water (machine type: 08.2205, TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany) and equilibrated with PBS. Then, the sample (500 µL of concentrated UF) was placed on the top of the filter of the column and immediately 20 fractions of 500 µL were collected.
EV were isolated from the purified and concentrated UF samples using a SEC method (Reshi et al. 2021 (link)). SEC columns were prepared by packing Econo-Pac® Chromatography columns (Bio-Rad, Hercules, CA USA, cat:7321010) with size exclusion chromatography resin (catalog. Sepharose 4 fast flow™, Cytvia, Uppsala, Sweden). In brief, the packed SEC columns were positioned vertically in a holder and washed by running through ultrapure Milli-Q® water (machine type: 08.2205, TKA Wasseraufbereitungssysteme GmbH, Niederelbert, Germany) and equilibrated with PBS. Then, the sample (500 µL of concentrated UF) was placed on the top of the filter of the column and immediately 20 fractions of 500 µL were collected.
10
Nanoparticle Characterization by NTA
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The number of particles in each fraction was determined using NTA. Briefly, particle concentrations in each of the fractions were measured with NTA—ZetaView® (PMX 110 V3.0 instrument by Particle Metrix GmbH, Inning am Ammersee, Germany) coupled with ZetaView NTA software for data analysis (Dissanayake et al. 2021a (link)). Operating instructions of the manufacturer were followed. For the auto-alignment of the instrument, 100 nm polystyrene particle size standards (Applied Microspheres B.V., Leusden, Netherlands, Catalogue no. 10100) were used. Samples were measured in the scatter mode for their total particle concentration and size profiles using the following settings: camera sensitivity 85, shutter 70, frame rate 30 frames per second (fps), number of cycles 3 and number of frames 11. Based on detectable quantities of nanoparticles during NTA measurements, the fractions with UF-EV were pooled per sample and further concentrated to 500 µL using Amicon® Ultra-2 mL centrifugal filters (Merck Millipore Ltd, Darmstadt, Germany, 10 KDs cut-off) at 2000 g for the subsequent characterization steps.
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