Cy3-(GT)6-SWCNTs and Cy3-(GT)30-SWCNTs were first filtered three times using 100 kDa Amicon centrifuge filters (Millipore) to remove free Cy3-DNA from solution. The cells were seeded onto 35 mm glass-bottom Petri dishes (MatTek) to a final concentration of 500 000 cells/dish and allowed to culture overnight in an incubator. The media was removed from each well, replaced with 1 mg/L of filtered Cy3-(GT)6-SWCNTs or Cy3-(GT)30-SWCNTs diluted in media and incubated for 30 min to allow internalization into the cells. The SWCNT-containing media was removed, the cells were rinsed three times with sterile PBS (Gibco), and fresh media was replenished for each sample. The Petri dishes were mounted in a stage incubator (Okolab) on an Olympus IX-73 inverted microscope with a UApo N 100×/1.49 oil immersion objective for epifluorescence imaging with a U-HGLGPS excitation source (Olympus) filtered through a Cy3 filter cube. The fluorescence images were analyzed by extracting average fluorescence intensity values of individual cell ROIs using ImageJ.
Amicon centrifuge filter
Manufactured by Merck Group
Sourced in Japan
Amicon centrifuge filters are filtration devices used to concentrate and purify macromolecules, such as proteins, enzymes, and nucleic acids, from complex solutions. They work by applying centrifugal force to the sample, which drives the liquid through a semi-permeable membrane, allowing the desired molecules to be retained while smaller components pass through.
Automatically generated - may contain errors
Lab products found in correlation
Vibra cell, by Sonics (4 mentions)
Uv vis nir spectrophotometer, by Jasco (4 mentions)
V 670, by Jasco (3 mentions)
Hiload 16 600 column, by GE Healthcare (3 mentions)
Synergy h1 hybrid reader, by Agilent Technologies (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (2 mentions)
Zetasizer nano z, by Malvern Panalytical (2 mentions)
Akta fast protein liquid chromatography system, by GE Healthcare (2 mentions)
Ix73 inverted microscope, by Olympus (1 mentions)
Sterile pbs, by Thermo Fisher Scientific (1 mentions)
Stage incubator, by Okolab (1 mentions)
Uapo n 100 1, by Olympus (1 mentions)
35 mm glass bottom petri dishes, by MatTek (1 mentions)
Bg nh2, by New England Biolabs (1 mentions)
Mwcnts, by Thermo Fisher Scientific (1 mentions)
Sodium deoxycholate solution, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Pbs buffer, by Thermo Fisher Scientific (1 mentions)
Centrifuge 5430 r, by Eppendorf (1 mentions)
23 protocols using amicon centrifuge filter
1
Quantifying Cellular Uptake of Cy3-SWCNTs
2
Fluorescent Dye Release from Nanoparticles
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For this assay, an Alexa Fluor 488 (AF488: Invitrogen, Carlsbad, CA) labeled version of BG was prepared. An amine modified version of BG (BG-NH2) was purchased from New England BioLabs, Inc. (Ipswich, MA) and modified with AF488 according to the manufacture’s instructions to produce AF 488 modified BG (BG-AF488). BG-488 was conjugated to NPCP as described above to produce (NPCP-(BG-AF488)). NPCP-(BG-AF488) (1 mg of Fe/mL) was diluted into PBS at pH 7.4 and acetate buffer at pH 5.0 containing 100 mM glutathione and incubated at 37 °C for 0, 1, 8, and 24 h. CP-(BG-AF488) was separated from NPCP-(BG-AF488) using Amicon centrifuge filters (30 000 MW cutoff, Millipore). Free CP-(BG-AF488) content in the filtrate was determined by fluorescence measurements. Percent CP-(BG-AF488) released from NPCP-(BG-AF488) was calculated using the fluorescence of total amount of drug released over 24 h.
3
Luteolin Inhibits S. mutans Amyloid Fibrillization
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Inhibition of S. mutans extracellular amyloid fibrillization by luteolin was tested by measuring the ThT fluorescence spectrum. For this, S. mutans strain ATCC 25175 was grown in a biofilm medium supplemented with 0.8% glucose (40 (link)) and cultured overnight at 37°C. After incubation, cells were removed by centrifugation at 7,000 g for 15 min. The supernatant containing extracellular proteins was filtered through a 0.2 μm Nalgene filter, concentrated with 10-kDa-cutoff Amicon centrifuge filters (Millipore) and equilibrated in PBS buffer, pH 7.2. Aliquots of extracellular proteins were stirred at 4°C for 60 h with 50 μg/mL luteolin. Following stirring, ThT was added to the samples to the final concentration of 2 μM, mixed, and incubated in the dark for 30 min at room temperature. Next, 200 μL of samples was transferred to a black-walled 96-well plate, excited at 442 nm, and fluorescent emission spectra were measured from 450 to 650 nm in a Synergy H1 hybrid reader (BioTek). A protein sample stirred in the absence of luteolin served as the positive control. A ThT-only sample was used as the negative control.
4
DNA-Stabilized Carbon Nanotube Dispersion
5
Functionalization of Carbon Nanomaterials
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MWCNTs (Alfa Aesar), RGO (Graphene Supermarket), and C60 (Alfa Aesar) were dispersed with (GT)15 DNA sequence in 1 mL of 100 mM NaCl, by adding 1 mg of raw nanomaterials to 2 mg of desalted DNA in a microcentrifuge tube. The mixtures were ultrasonicated using a 1/8” tapered microtip for 30 minutes (Only the C60 solution was ultrasonicated for 90 minutes) at 40% amplitude, with an average power output of 8 W, in a 0 °C temperature-controlled microcentrifuge tube holder. The free DNA was removed using 100 kDa Amicon centrifuge filters (Millipore), and the DNA functionalized carbon nanomaterials pellet was resuspended in DI water.
6
Fluorescence Monitoring of Cy3-DNA-SWCNTs
7
Dispersing Carbon Nanotubes with DNA
8
DNA-Nanotube Complexation and Purification
9
DNA-Wrapped Single-Walled Carbon Nanotube Preparation
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SWCNTs produced by the HiPco process (Nanointegris) were used throughout the study. SWCNTs were dispersed with single-stranded DNA oligonucleotides in 1 mL of 100 mM NaCl (Sigma-Aldrich), by adding 1 mg of raw nanotubes to 2 mg of desalted (GT)n (n = 3, 6, 9, 12, 15, or 30), (CT)15, or C30 oligonucleotide (Integrated DNA Technologies) in a microcentrifuge tube. The mixtures were ultrasonicated using a 1/8” tapered microtip (Sonics Vibracell; Sonics & Materials) for 30 minutes at 40% amplitude, with an average power output of 8 W, in a 0 °C temperature-controlled microcentrifuge tube holder. After sonication, the dispersion was ultracentrifuged (Sorvall Discovery M120 SE) for 30 minutes at 250,000 g, and the top 80% of the supernatant was extracted. The concentration was determined with a UV/vis/NIR spectrophotometer (Jasco, Tokyo, Japan), using the extinction coefficient of A910 = 0.0255455 (link) L.mg−1.cm−1. The free DNA was removed using 100 kDa Amicon centrifuge filters (Millipore). For each sample, filtration was repeated 4 times, and the DNA-SWCNTs pellet was resuspended in DI water in each step including the final step.
10
Luteolin Inhibits S. mutans Amyloid Fibrillization
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Inhibition of S. mutans extracellular amyloid fibrillization by luteolin was tested by measuring the ThT fluorescence spectrum. For this, S. mutans strain ATCC 25175 was grown in a biofilm medium supplemented with 0.8% glucose (40 (link)) and cultured overnight at 37°C. After incubation, cells were removed by centrifugation at 7,000 g for 15 min. The supernatant containing extracellular proteins was filtered through a 0.2 μm Nalgene filter, concentrated with 10-kDa-cutoff Amicon centrifuge filters (Millipore) and equilibrated in PBS buffer, pH 7.2. Aliquots of extracellular proteins were stirred at 4°C for 60 h with 50 μg/mL luteolin. Following stirring, ThT was added to the samples to the final concentration of 2 μM, mixed, and incubated in the dark for 30 min at room temperature. Next, 200 μL of samples was transferred to a black-walled 96-well plate, excited at 442 nm, and fluorescent emission spectra were measured from 450 to 650 nm in a Synergy H1 hybrid reader (BioTek). A protein sample stirred in the absence of luteolin served as the positive control. A ThT-only sample was used as the negative control.
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