DNA was synthesized at a 1 µmole scale of solid phase synthesis on an ABI 392 DNA synthesizer (Applied Biosystems) using materials purchased from Glen Research (Sterling, VA) or Chemgenes (Wilmington, MA). DNA was deprotected accordingly to the manufacturer’s instructions, and purified by reverse phase HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, Waltham, MA). Dimethoxytrityl (DMT) protecting group on DNA was removed using 0.5 M acetic acid. DNA was desalted and quantified on a Genesys 10 s UV-Vis spectrometer (Thermo Fisher Scientific, Waltham, MA). Fluorophores, thiol, or HEG were modified following the manufacturer’s instructions. All DNAs, including HEG linkers, had phosphorothioate backbone. DNA modified with Alexa488 or Alexa555 were from Integrated DNA Technology (Coralville, IA).
Alexa 488
Manufactured by Integrated DNA Technologies
Sourced in United States
Alexa-488 is a fluorescent dye used for labeling and detecting biomolecules. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, making it suitable for applications that use green fluorescent detection.
Automatically generated - may contain errors
Lab products found in correlation
Abi 392 dna synthesizer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Genesys 10 s uv vis spectrometer, by Thermo Fisher Scientific (1 mentions)
Novocyte cytometer, by Agilent Technologies (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Trichloroethylene, by Merck Group (1 mentions)
Acetone, by Merck Group (1 mentions)
Rq1 rnase free dnase buffer, by Promega (1 mentions)
Cell culture media and reagents, by Thermo Fisher Scientific (1 mentions)
Celltiter blue reagent, by Promega (1 mentions)
Dnase, by Promega (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Nuclease protease free water, by Quality Biological (1 mentions)
Dna oligonucleotide, by Integrated DNA Technologies (1 mentions)
7 protocols using alexa 488
1
Synthesis and Purification of Modified DNA
2
Cellular Uptake of DNA/RNA Nanocubes
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PBMCs were either left untreated or incubated in the presence of DNA and RNA cubes alone, complexed with Lipofectamine, or complexed with G5 amine-terminated dendrimers. After 24 h of incubation, the cells were washed to remove the excess particles, reconstituted in the flow cytometry buffer, and analyzed using a Novocyte cytometer (ACEA Biosciences, San Diego, CA, USA). All cubes used for experiments were labeled with Alexa 488 (Integrated DNA technologies, Coralville, IA, USA ). The final particle concentration was 10 nM, the same as was used in the cytokine experiments. The cells were separated according to their forward and side scatter, and the live populations of lymphocytes and monocytes were gated into the green fluorescent channel for the detection of particle uptake. The data analysis was performed using the FCS Express software (DeNovo Software Inc., Pasadena, CA, USA).
3
Aptamers Binding Affinity for HSC-T6 Cells
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Two aptamers (aptamer 6 and aptamer 9), which showed more similar sequences with other aptamers in the SELEX, were selected for the affinity assay. A sticky bridge sequence (5'-CACAAGGAACAAGG-3') was added to the aptamers either at 5' or at 3' end. A complementary sequence labeled with Alexa-488 (Integrated DNA Technologies, Coralville, IA) was annealed with the sticky bridge of the aptamer. Binding affinities of these aptamers to HSC-T6 cells were determined as previously reported 8 (link). Briefly, the cells were detached using non-enzymatic cell dissociation solution and re-suspended in Opti-MEM medium. Alexa-488 labeled aptamers (100 nM) were incubated with HSC-T6 cells at 37 oC for 1 h with gentle rotation. The cells were washed three times with PBS and then subjected to fluorescence analysis on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ) using an excitation wavelength of 488 nm with a 535 nm emission filter. Cellular uptake of other aptamers was also determined in rat HSC-T6 cells using flow cytometry. The apparent Kd value of aptamers that exhibited high binding affinity to HSC-T6 cells were determined in LX-2 and HSC-T6 cells. The results were calculated by non-linear fit in Graphpad Prism.
4
Fluorescent Labeling of miRNA Molecules
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Monocrystalline silicon wafer diced in 1 cm × 1 cm substrates (FBK Microfabrication Facility) and fluorescamine (4′-phenylspiro (2-benzofuran-3,2′-furano)-1,3′-dione) were purchased from Thermo Scientific (Waltham, MA, United States). Trichloroethylene, acetone, and all powders for buffer solutions were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). Synthetic hsa-miR-21 conjugated at the 5′ end with the fluorescent dye Alexa-488 (5′-Alexa488-UAGCUUAUCAGACUGAUGUUGA-3′; miR − 21) and synthetic hsa-miR-16 conjugated at the 5′ with the same dye (5′-UAGCAGCACGUAAAUAUUGGCG-3′) were synthetized by Integrated DNA Technologies (IDT, Leuven, Belgium).
5
Characterization of DOPE-based Lipid Nanoparticles
6
Visualizing CpG-Hemozoin Binding in Cells
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Five microgram of human CpG-2006 DNA with a phosphorothionate (PTO) backbone bound to Alexa-488 (Integrated DNA Technologies, Coralville, USA) were mixed with 100 µg/ml of sonicated synthetic hemozoin [32] (link)and co-incubated with rocking for 2 h followed by washing of the complex three times with PBS. Bound and unbound DNA were determined by measuring the DNA concentration in the collected supernatant, using the Nanodrop. BL2 and IM 171 cells were each plated at 1 ml (with approximately 500,000 cells per confocal plate). The CpG-Alexa-488-hemozoin complex was added to the cell lines BL2 and SP-IM 171, and incubated for 2 hours after which confocal microscopy was carried out. Confocal reflection microscopy for detection of hemozoin was combined with fluorescence microscopy to detect the Alexa-488 tagged CpG on a Leica SP2 AOBS confocal laser-scanning microscope as described in detail in [54] (link).
7
Dye-free DNA Block Structure Assembly
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