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Transfection efficiency in live cell imaging system

Updated: Jan 2

Transfection efficiency is the measurement of cells transfected which is affected by factors that may inhibit cells from expressing the transfected protein such as cell cycle progression, circadian rhythm of gene expression activity, and promotor activity [1].

What is transfection and its purpose

The transfection word itself originally refers to cellular infection with isolated viral nucleic acid producing the complete virus in the cell or new phage particles [2][3]. Today, the term is used as a technique to insert foreign nucleic acid (DNA or RNA) into a cell to alter the properties of the cell [3].

The main purpose of transfection is to study the function of genes or protein expression, by enhancing or inhibiting specific gene expression [4].

Where is transfection used?

Transfection is a powerful tool enabling the various studies and experiments such as

1. Gene functions and gene products in cells by analyzing gene expression such as DNA, RNA, or proteins or by knockdown method targeting RNA or protein level.

2. Transcript expression to analyze the protein level of transfected mRNA.

3. Drug screening is to evaluate the cytotoxicity using the transfection reagent to choose the optimal drug or to study the mechanism of drug.

4. Cancer research to study the effect of genes on tumor growth and progression.

5. Virus production to study viral replication or for vaccine development.

How to analyze transfection efficiency in live cell imaging system

Transfection efficiency is calculated using this simple calculation.

Transfection efficiency(%) = (Number of fluorescent [transfected] cells / total number of cells)x100.

Thus, higher transfection efficiency is more desirable for researchers using reporter genes to study transfection processes and conditions with fluorescent proteins such as green fluorescent proteins (GFP) or red fluorescent protein (RFP). When the transfection is done successfully, cells that express the target protein are easily distinguishable because of the intracellular fluorescent protein. This process is simply done using a live cell imaging system [5].

This is a time-lapse of transfection efficiency taken by JuLI Stage. Over time, the fluorescence level increases, in this case RFP has been used.


1. Sandbichler, Adolf Michael, et al. “A method to evaluate the efficiency of transfection reagents in an adherent zebrafish cell line.” BioResearch Open Access, vol. 2, no. 1, 2013, pp. 20–27,

2. “Transfection Definition & Meaning.” Merriam-Webster, Merriam-Webster, Accessed 22 Sept. 2023.

3. “Transfection.” Encyclopædia Britannica, Encyclopædia Britannica, inc., Accessed 22 Sept. 2023.

4. Kim, Tae Kyung, and James H Eberwine. “Mammalian Cell Transfection: The Present and the Future.” Analytical and Bioanalytical Chemistry, U.S. National Library of Medicine, Aug. 2010,

5. Lin Peng, Wendian Xiong, Yanfei Cai, Yun Chen, Yang He, Jianfeng Yang, Jian Jin & Huazhong Li (2017) A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye, Bioengineered, 8:3, 225-231, DOI: 10.1080/21655979.2016.1222995

Objective lens

4x, 10x, 20x

Channel type

Brightfield, GFP, RFP, DAPI

Vessel type

Well plate, flask, dish, slide

Incubator installation


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