Methods for delivering compounds into a cell
a cell and compound technology, applied in the field of intracellular delivery, can solve the problems of high cost, low efficiency of transfection (delivery of genetic material into cells) and subsequent gene expression, and difficulty in gene expression
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
Effect of Ultrasound on the Temperature of an Aqueous-Based Medium in Culture Plate Well Phantoms and on Cell Viability
The first phase of evaluating the effect of ultrasound was to measure the amount of heating caused by the ultrasound energy. The experimental protocol was designed to evaluate the heating in an individual well of a 6 well culture plate while exposed to ultrasound. Ultrasound was applied for 30 seconds to each well and the relationship between energy and heating is shown in Table 2 below.
TABLE 2Temperature increaseTemperature increaseEnergyat 10% Duty Cycleat 100% Duty Cycle0.5 W / cm2 0° C.0.5° C.1 W / cm20° C.1.5° C.2 W / cm20.5° C. 2.9° C.
A follow-up experiment was carried out to assay the cell viability after ultrasound exposure. A cell proliferation kit using sodium 3′-[1-(phenylamino-carbonyl)3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT, Boehringer-Mannheim, Indianapolis, Ind.) as the cell viability indicator was carried out to eva...
example 2
Effect of Ultrasound on Gene Expression in Cell Culture
Materials and Methods for Transfection and Measurement of Gene Expression
The DNA plasmid used was pCAT Control (GenBank accession number X65321) (Promega, Madison, Wis.) (see FIG. 3).
The plasmid was transformed into DH5-αEscherichia coli competent cells (Life Technologies, Gaithersburg, Md.). The cells were plated on LB agar plates (Bio 101, Vista, Calif.) that contained ampicillin (Boehringer Mannheim Biochemicals (BMB), Indianapolis, Ind.). Resistant colonies were selected, grown up and a Wizard mini-prep (Promega) plasmid DNA extraction was carried out. Plasmid DNA was cut with restriction enzyme EcoRI (BMB) and run on a 1% agarose gel (BMB). After fragments were evaluated for size, the remaining culture was used to start a large culture and a Wizard maxi-prep was carried out to produce DNA for transfection. The DNA was quantified using a Hoefer TKO-100 mini-DNA fluorometer. The DNA was then ready for use in transfectio...
example 3
Application Using DNA with a Lipid Carrier and a Cavitator
The cells and the DNA / lipid complex were prepared as in Example 2. Six well plates were seeded with HeLa cells and filled with 16 ml of media as in Example 2. The lipid added was increased to 135 μg to allow for the increase in volume and the DNA was also increased to 22.5 μg per well. One hour after the complex was added, 100 μl of a liposome comprised of the lipids dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine coupled to polyethylene glycol 5000 (DPPE-PEG5000), and dipalmitoylphosphatidic acid (DPPA), in a ratio of about 82%:8%: 10% (mole %) was added to each well. The DPPE-PEG5000 was comprised of DPPE and PEG5000 in a ratio of about 20%:80% (weight %). PEG5000 refers to PEG having an average molecular weight of about 5000. In addition, a negative control was added which included cells grown up not transfected with CAT / lipid complex and without ultrasound treatment.
The six well plate was th...
PUM
| Property | Measurement | Unit |
|---|---|---|
| frequency | aaaaa | aaaaa |
| pressure | aaaaa | aaaaa |
| speed | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More