The liposome structures were examined by the FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). A 200-mesh copper grid-supported holey carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) was glow-discharged in an argon/oxygen atmosphere for 15 s. Four microliters of the liposome sample containing ~0.5 mM lipids were pipetted onto the copper grid, paper-blotted for 3 s in a 100% humidified chamber at 4 °C and plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot system (FEI, Hillsboro, OR, USA). The grids were stored in liquid nitrogen until mounted for imaging. EM imaging was conducted in bright-field mode at an operating voltage of 200 kV. Images were recorded at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Å2) with a 4k 4k charge-coupled device camera (Glatan, Pleasanton, CA, USA) at a magnification of 50,000×. All experiments were carried out at the Academia Sinica Cryo-EM Facility (Taipei, Taiwan).
Hc200 cu
Manufactured by Electron Microscopy Sciences
Sourced in United States
The HC200-Cu is a high-contrast grid holder for transmission electron microscopy (TEM). It is designed to securely hold copper grids for sample observation and analysis in a TEM instrument.
Automatically generated - may contain errors
Lab products found in correlation
Fei tecnai g2 f20 twin tem, by Thermo Fisher Scientific (2 mentions)
Vitrobot system, by Thermo Fisher Scientific (1 mentions)
2100plus, by JEOL (1 mentions)
Cf200 cu, by Electron Microscopy Sciences (1 mentions)
Orius sc1000 camera, by Ametek (1 mentions)
4k charge coupled device camera, by Ametek (1 mentions)
Tecnai g2 f20 twin tem, by Thermo Fisher Scientific (1 mentions)
Tecnai f20, by Thermo Fisher Scientific (1 mentions)
4 protocols using hc200 cu
1
Cryo-EM Imaging of Liposome Structures
2
Transmission Electron Microscopy Sample Preparation
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Copper grids (CF200-Cu, Electron Microscopy
Sciences) and holey carbon grids (HC200-Cu, Electron Microscopy Sciences)
were plasma treated (15 s, O2/H2) on a Gatan
Solaris plasma cleaner. Samples for TEM were prepared by adding a
4 μL sample to the grid and incubating for 30 s, after which
excess solution was blotted off with filter paper, and the sample
was dried overnight. Samples for cryo-TEM were prepared with a Leica
EM GP automatic plunge freezer. Samples (4 μL) were added to
a grid in an environmental chamber (relative humidity 90%, temperature
20 °C). Excess suspension was blotted on filter paper, and the
obtained film was vitrified in liquid ethane. Samples were stored
in liquid nitrogen until the day of use.
TEM samples were imaged
on a JEOL 2100F or JEOL 2100Plus using 200 kV, while cryo-TEM samples
were imaged only on the JEOL 2100Plus using 200 kV with the minimum
dose system. Cryo-TEM samples were imaged at −170 °C on
a Gatan914 cryo-holder for cryo-TEM imaging. Micrographs were taken
using the Gatan Orius SC1000 camera at a magnification of either 30 000×
or 15 000×.
Sciences) and holey carbon grids (HC200-Cu, Electron Microscopy Sciences)
were plasma treated (15 s, O2/H2) on a Gatan
Solaris plasma cleaner. Samples for TEM were prepared by adding a
4 μL sample to the grid and incubating for 30 s, after which
excess solution was blotted off with filter paper, and the sample
was dried overnight. Samples for cryo-TEM were prepared with a Leica
EM GP automatic plunge freezer. Samples (4 μL) were added to
a grid in an environmental chamber (relative humidity 90%, temperature
20 °C). Excess suspension was blotted on filter paper, and the
obtained film was vitrified in liquid ethane. Samples were stored
in liquid nitrogen until the day of use.
TEM samples were imaged
on a JEOL 2100F or JEOL 2100Plus using 200 kV, while cryo-TEM samples
were imaged only on the JEOL 2100Plus using 200 kV with the minimum
dose system. Cryo-TEM samples were imaged at −170 °C on
a Gatan914 cryo-holder for cryo-TEM imaging. Micrographs were taken
using the Gatan Orius SC1000 camera at a magnification of either 30 000×
or 15 000×.
3
Cryo-EM Analysis of Dox-MB Fragments
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The structure of fragment species of Dox-MBs was observed using the cryo-EM imaging performed on the FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). Initially, a 200-mesh copper grid-supported holey carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA) was placed in an argon/oxygen atmosphere and was surface-modified by glow-discharge for 15 s. A 4 μl sample was added onto the copper grid blotted for 3 s in a 100 % humidified chamber at 4 °C. Subsequently, it was flash-frozen in liquid ethane that had been cooled by liquid nitrogen using the Vitrobot sample plunger system (FEI, Hillsboro, OR, USA). The prepared copper grid was stored in liquid nitrogen until imaging. Imaging was conducted in the bright-field mode for cryo-EM with an operating voltage of 200 kV. The images were captured at a magnification of 50,000 × with a 4 k × 4 k charge-coupled device camera (Gatan, Pleasanton, CA, USA).
To investigate the size distribution of fragment species in LDox-MBs and HDox-MBs, the samples with and without US were centrifuged, and the bottom layer solution was analyzed. Particle size and concentration were measured using dynamic light scattering (DLS, model ZetaSizer 3000, Malvern, Worcs., UK). Before analysis, the bottom layer solution was diluted 1000-fold with PBS to achieve concentrations between 106 and 109 particles/ml.
To investigate the size distribution of fragment species in LDox-MBs and HDox-MBs, the samples with and without US were centrifuged, and the bottom layer solution was analyzed. Particle size and concentration were measured using dynamic light scattering (DLS, model ZetaSizer 3000, Malvern, Worcs., UK). Before analysis, the bottom layer solution was diluted 1000-fold with PBS to achieve concentrations between 106 and 109 particles/ml.
4
Cryo-EM Structures of LNC-Dox
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The structures of LNC-Dox were examined using an FEI Tecnai G2 F20 TWIN TEM (FEI, Hillsboro, OR, USA). Samples were prepared for cryo-EM on an FEI Vitrobot by pipetting 4 μL of the reaction mixture containing ~0.5 mM lipids onto a 200-mesh perforated carbon film (HC200-Cu, Electron Microscopy Sciences, Hatfield, PA, USA), blotted for 3 s, and stored in liquid nitrogen until imaging. Images were taken at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Å2) at 50,000-fold magnification on a FEI Tecnai F20, operating at 200 kV. All experiments were carried out at the Academia Sinica Cryo-EM Facility (Taipei, Taiwan).
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