Measurement of the Transfection Efficiency of Cultivated Cells

Capturing clear images without fluorescence blurring

Transfection is the process of introducing nucleic acids into animal cells in order to make cells introduce specific genes to express a protein of interest. Through this process, transfection efficiency can be analyzed, making it vital for current medical research.

The success or failure of a gene transfer relies on the optimization of the transfer conditions. As such, various researchers have repeatedly carried out trial and error in search of the optimal process.

There are a wide range of processes to accomplish this, but they can generally be classified into the following three types: (1) chemical processes, (2) biological processes, and (3) physical processes. Each process has its strengths and weaknesses, so it is necessary to select the appropriate process to match the needs of the experiment and cell type.

After selecting the optimal process, preparation for the soundness and survival rate of the cell system, the cell density, and other components are required to ensure that the transfection is successful before starting the experiment. Although it is extremely important to accurately measure the transfection efficiency, many researchers can struggle in doing so.

One method for measuring the transfection efficiency is to use a fluorescence microscope. This can be done by counting the total number of observed cells and the number of cells that express fluorescence and then scoring these values. However, when using conventional fluorescence microscopes, this approach posed various problems and was often very difficult.

First, in order to observe the fluorescence, the specimen had to be taken to a darkroom to perform the experiment, which made the process challenging. Additionally, it was difficult to extract the outlines of each cell, resulting in inaccurate cell counts.

Photobleaching due to fluorescence observation was a headache for many researchers. The health of the cells is extremely important, so a method that does not weaken the cells was required.

In addition, 3rd party software was required to count and analyze the number of cells, which oftentimes did not integrate well with the microscope being used and was complicated to operate.

Counting cells using a phase contrast image
Counting cells using a phase contrast image
Counting expression events (fluorescent points) using cells as mask areas
Counting expression events (fluorescent points) using cells as mask areas
Counting expression events (fluorescent points) using cells as mask areas
Cell
903
Expression
89
Efficiency
9.9%

Objective lens: CFI Plan Fluor DL 10x

Using the All-in-One Fluorescence Microscope BZ-X800