GFP fluorescent proteins were fused to the N-terminus of AtUBL5a and AtUBL5b to investigate the localization of GFP-AtUBL5a and GFP-AtUBL5b, respectively. Gold particles (1.0 μm; Bio-Rad, Richmond, CA) coated with DNA (0.1 μg/μL) were delivered into onion epidermal cells using the Biolistic® PDS-1000/He system (Bio-Rad, Richmond, CA) with 1,100 psi rupture discs. After incubation at 22°C for 16 hours, GFP fluorescent signals in the onion epidermal cells were recorded under an epifluorescence microscope (AxioImager Z1, Carl Zeiss) equipped with a CCD camera (AxioCam HRc, Carl Zeiss). The empty vector pA7-GFP was used a as control. Images were processed using Spot Advance and Adobe Photoshop software.
Gold particles
Manufactured by Bio-Rad
Sourced in United States
Gold particles are spherical nanoparticles made of gold. They have a core function of enabling the visualization and detection of biological molecules and structures in various scientific and medical applications.
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Lab products found in correlation
Helios gene gun, by Bio-Rad (12 mentions)
Biolistic pds 1000 he particle delivery system, by Bio-Rad (8 mentions)
Pds 1000 he, by Bio-Rad (5 mentions)
Helium pressurized gun, by Bio-Rad (4 mentions)
Helios gene gun system, by Bio-Rad (4 mentions)
Biolistic pds 1000 he system, by Bio-Rad (3 mentions)
Amigo2 ddk, by OriGene (2 mentions)
Helios gene gun system instruction manual, by Bio-Rad (2 mentions)
Tefzel tubing, by Bio-Rad (2 mentions)
D luciferin, by Fujifilm (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Axio imager z1, by Zeiss (1 mentions)
Model pds 1000 he, by Bio-Rad (1 mentions)
Cm dii, by Merck Group (1 mentions)
S9883, by Merck Group (1 mentions)
1 μm gold particles, by Bio-Rad (1 mentions)
Bx53 microscope system, by Olympus (1 mentions)
Axioscope, by Zeiss (1 mentions)
Axiovert 100 microscope, by Zeiss (1 mentions)
58 protocols using gold particles
1
Localization of GFP-tagged AtUBL5 proteins
2
Gold Particle Preparation Protocol
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Sixty milligrams of gold particles (0.6 µm, Biorad® Hercules, CA. USA) were prepared according to Daniell et al. (2005).
3
Transient Expression Protocols in N. benthamiana
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For agroinfiltration, Agrobacterium EHA105 strain harboring the tested expression construct(s) was grown in LB medium supplemented with spectinomycin (100 μg/ml) overnight at 28°C. Cells were harvested by centrifugation and resuspended to optical density of A600 = 0.1 in infiltration buffer [10 mM MgCl2, 10 mM MES (pH 5.5), 100 μM acetosyringone]. Bacterial suspension was incubated for 2 h at room temperature and infiltrated into the abaxial sides of 3- to 4-week-old intact N. benthamiana leaves with a 1-ml needleless syringe. Plants were grown for 48–72 h under standard growth conditions before being harvested.
For biolistic delivery, DNA preparations of the tested constructs were mixed at a 1:1 w/w ratio, and 100 μg DNA was adsorbed onto 10 mg of 1-μm gold particles (Bio-Rad, Hercules, CA). These microprojectiles were bombarded into the leaf epidermis of N. benthamiana using a portable Helios gene gun system (Model PDS-1000/He, Bio-Rad) at a pressure of 90–150 psi, and tissues were analyzed 48 h after microbombardment.
For biolistic delivery, DNA preparations of the tested constructs were mixed at a 1:1 w/w ratio, and 100 μg DNA was adsorbed onto 10 mg of 1-μm gold particles (Bio-Rad, Hercules, CA). These microprojectiles were bombarded into the leaf epidermis of N. benthamiana using a portable Helios gene gun system (Model PDS-1000/He, Bio-Rad) at a pressure of 90–150 psi, and tissues were analyzed 48 h after microbombardment.
4
Sparse Neuronal Transfection in Retina
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We coated gold particles (diameter: 1.6 μm, Bio-Rad) with plasmids encoding cytosolic tdTomato and postsynaptic
density protein 95 (PSD95) fused at its C terminus to YFP (Kerschensteiner et al.,
2009 ), or AMIGO2-DDK (Origene). We used a helium-pressurized gun (40 psi, Bio-Rad) to deliver particles to a sparse
population of cells in the ganglion cell layer and incubated the transfected retinas in mACSFHEPES in a humid
oxygenated chamber at 33°C for 16–18 hr (Morgan and Kerschensteiner,
2011 ). We identified ON SACs by their characteristic arbor morphology.
density protein 95 (PSD95) fused at its C terminus to YFP (
2009
population of cells in the ganglion cell layer and incubated the transfected retinas in mACSFHEPES in a humid
oxygenated chamber at 33°C for 16–18 hr (
2011
5
Biolistic Delivery of Fluorescent Plasmids to Retinas
6
DNA Delivery via Gold Particle Microcarriers
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Gold particles (Bio-Rad Laboratories, Hercules, CA 94547, USA) were used as microcarriers for the high-helium-pressure delivery of DNA vaccination. Microcarrier loading quantity (MLQ), the amount of Gold particles, was set to 0.5 mg per target according to the manufacturer’s recommendation for in vivo delivery. The amount of DNA loaded per mg of microcarriers is referred to as the DNA loading ratio (DLR), was set to 1 μg/mg DNA/Gold particles, giving 0.5 mg of Gold particles and 0.5 μg of DNA per cartridge. Cartridge preparation was conducted according to the manufacturer’s protocol (BIO-RAD Helios gene gun system instruction manual, #165–2431 and #165–243). Briefly, spermidine, CaCl2, polyvinylpyrrolidone solutions and Gold particles were added to the purified DNA (at a concentration of 500 ng/μl) to bind it to the Gold particles. The DNA-gold mixture was injected to plastic tubes and dried using a gentle stream of nitrogen gas. Gold-DNA coated tubes were cut into 1 cm-long cartridges. Cartridges were stored in a desiccator at 4 °C until use.
7
Neuronal Morphology Analysis of Hippocampal CA1
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To analyze the morphology of dendrites and the spines of hippocampal CA1 neurons from 5-month-old mice, the neurons were marked with the fluorescent dye, 1-1’-Dioctadecyl-3,3,3,’3’-tetramethylindocarbocyanine perchlorate (DiI; CM-DiI; Sigma-Aldrich, St. Louis, MO, USA) as previously described.18 (link) Briefly, 2 mg of DiI was fully mixed with 8 mg of 1.0-µm gold particles (BioRad, Hercules, CA, USA). Given that DiI is soluble in organic solvents, after 300 µL of methylene chloride or dimethylformamide was added to it, a solution was formed. Thus, DiI adhered to the surface of the gold particles. Thereafter, the DiI-gold particle warhead was placed in a gene gun, and the particles were scattered on the surface of the brain slices using helium (150–180 p.s.i.). This was followed by rinsing of the brain slices twice with 0.1 M PBS. Finally, the brain slices were stored in 0.1 M PBS and incubated in a 4°C refrigerator for 12–24 h.
8
Microparticle Bombardment for Phaeodactylum Transformation
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Phaeodactylum tricornutum cells (1.5 × 108 total) were collected from exponentially growing cultures and spread onto 1% agar plates containing F/2 medium with 20 g L−1 sea salt (Sigma S9883). Transformations were carried out 24 h later using the microparticle bombardment method adapted from (Apt et al., 1996 (link)) with minor modifications as follows. Gold particles (0.6 µm diameter, BioRad) were coated with DNA using 1.25 M CaCl2 and 20 mM spermidine. As a negative control, beads were coated with 5 µg NAT selection plasmid and 5 µg empty vector. For each polycistronic cassettes (NAT-T2A-GUS, GUS-T2A-NAT, NAT-P2A-GUS, or GUS-P2A-NAT), beads were coated with 5 µg of DNA. A burst pressure of 1,550 psi and a vacuum of 25 Hg were used.
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Phaeodactylum tricornutum cells (1.5 × 108 total) were collected from exponentially growing cultures and spread onto 1% agar plates containing F/2 medium with 20 g L−1 sea salt (Sigma S9883). Transformations were carried out 24 h later using the microparticle bombardment method adapted from (Apt et al., 1996 (link)) with minor modifications as follows. Gold particles (0.6 µm diameter, BioRad) were coated with DNA using 1.25 M CaCl2 and 20 mM spermidine. As a negative control, beads were coated with 5 µg NAT selection plasmid and 5 µg empty vector. For each polycistronic cassettes (NAT-T2A-GUS, GUS-T2A-NAT, NAT-P2A-GUS, or GUS-P2A-NAT), beads were coated with 5 µg of DNA. A burst pressure of 1,550 psi and a vacuum of 25 Hg were used.
9
Gold Particle-Based DNA Coating Protocol
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Plasmid DNA was coated onto 1 μm gold particles (Bio-Rad, Hercules, CA, USA) by the procedure recommended by the producer of gold particles. Each cartridge contained 1 μg DNA coated onto 0.5 mg of gold particles.
10
Transient Expression of Fluorescent Proteins in Onion Cells
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Constructs encoding the nEYFP-fused proteins, cEYFP-fused proteins, and DsRed1 were introduced into onion epidermal cells by particle bombardment with the PDS-1000 He Biolistic Particle Delivery System (Bio-Rad, CA, USA). Gold particles (0.6 mg and 1.6 μm in diameter; Bio-Rad, CA, USA) were coated with 0.5 μg of each vector for each bombardment. The plasmid-coated particles were bombarded at 1,100 psi and 9-cm distance. The bombarded cells were incubated at 25 °C in darkness for 24 h. YFP and DsRed1 fluorescence was visualized using the BX53 microscope system (Olympus, Japan). The U-FBNA filter cube (excitation filter, BP470–495 nm; dichromatic mirror, DM505 nm; and suppression filter, BA510–550 nm) and U-FGW cube (excitation filter, BP530–550 nm; dichromatic mirror, DM570 nm; and suppression filter, BA575IF nm) were used for detecting YFP and DsRed1 fluorescence, respectively.
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