Introduction to transfection
Transfection is a process involving the introduction of foreign nucleic acids into eukaryotic cells, resulting in modification of host cell genome. The modification can take place either through the expression of exogenous genetic material or by means of endogenous gene silencing. Transfection is a powerful tool for studying cell physiology and complex molecular mechanisms of diseases, as it can shed more light on gene regulation and protein function1. Various types of nucleic acids can be transfected into mammalian cells, including plasmid DNA (pDNA) and small, non-coding RNAs, such as small interfering RNA (siRNA), microRNA (miRNA), and short hairpin RNA (shRNA)2. Intact functional proteins (e.g. Cas9, recombinant proteins, antibodies) and peptides can also be directly delivered into cells, offering significant potential for clinical applications3. Broadly, the transfection methods can be subdivided into biological (e.g. transduction), physical (e.g. electroporation, microinjection), and chemical (e.g. lipofection, calcium phosphate transfection) methods1.
Evaluation of transfection efficiency
Transfection efficiency is the proportion of cells in a sample that acquired a foreign element and it can be assessed using a number of different approaches. Firstly, transfection efficiency can be confirmed and quantified through the analysis of the target gene or protein expression post-transfection. This method evaluates total gene/protein expression from a transfected cell population and it can be measured through e.g. western blotting, real-time quantitative PCR (real-time qPCR), reporter system, or molecular imaging. Transfection efficiency can also be evaluated by determining the proportion of transfected cells within a total cell population. This can be done by coupling a transfected gene with a fluorescent reporter (see: Lux3 FL & Exact FL compatible fluorescent protein list), either by placing them on separate delivery vectors or by introducing them together, through a transcriptional/translational reporter gene fusion4. Subsequently, transfection efficiency can be assessed by estimating the number of fluorescent cells either using flow cytometry or fluorescence microscopy5.
The CytoSMART Lux3 FL: real-time analysis of transfection efficiency
Live-cell, time-lapse imaging is a powerful approach that can be used for monitoring the time course of transfection, providing valuable, time-dependent insight into the process6. Compared to other methods of transfection efficiency analysis (i.e. western blotting, flow cytometry, or real-time qPCR), live-cell imaging and microscopy, in general, offer a more straightforward and direct route to evaluating the efficiency of foreign molecule uptake. However, even among different live-cell imaging modalities, there are approaches capable of providing reliable results quickly and easily, while requiring minimal expenses.
The CytoSMART Lux3 FL combines brightfield and fluorescence imaging to directly visualize and monitor the uptake and expression of foreign elements by transfected cells (Figure 1). The device can also automatically evaluate the efficiency of transfection based on the fluorescence surface area or the number of fluorescent objects in the image:
- Surface area: transfection efficiency can be calculated as a ratio of the surface area occupied by fluorescent cells to the total surface area of cells, as determined by the brightfield channel (Figure 2)
- Object count: while transfection efficiency cannot be measured directly using the integrated Object Count algorithm, the CytoSMART Lux3 FL can calculate the number of fluorescent objects in the image and track fluorescent object number over time (Figure 3)
Monitoring the process of transfection not only allows to examine transfection-induced changes in gene expression and notice trends that otherwise might have been missed, but it can also assist in the optimization of a transfection protocol. Viability and density (confluency) of cells are known factors that influence transfection efficiency. Live-cell imaging can be used to assess cell health and confluency prior to and during transfection to maximize the efficiency of nucleic acid uptake.
Figure 1 | Time-lapse video of HepG2 cells transduced using the BacMam System. HepG2 cells were transduced in suspension using CellLight Nucleus-RFP BacMam 2.0 and CellLight Actin-GFP BacMam 2.0 reagents for fluorescent detection of nuclei (red signals) and filamentous actin (green signals). Images were taken using the CytoSMART Lux3 FL with a snapshot interval of 30 min.
Figure 2 | Cell coverage graph. HepG2 cells were cultured on tissue culture plastic and transduced with BacMam-Actin-GFP and BacMam-Nucleus-RFP constructs to visualize expression of actin filaments and nuclei, respectively. The total cell coverage over time is depicted as the blue line, the green fluorescent coverage (actin filaments) as the green line and the red fluorescent coverage (nuclei) as the red line.
Figure 3 | Object count graph. The graph displays the number of fluorescent objects detected in the image. Red fluorescent objects represent the expression of BacMam-Actin-GFP and green fluorescent objects represent the expression of BacMam-Nucleus-RFP within HepG2 cells.
The CytoSMART Exact FL: endpoint analysis of transfection efficiency
The CytoSMART Exact FL is an automated, dual fluorescence cell counter with an expanded field of view and increased optical resolution that provides accurate and reliable analysis of transfection efficiency. Similar to other microscopy-based approaches, the Exact FL directly visualizes transfected, fluorescently-labeled cells in a sample. However, unlike the CytoSMART Lux3 FL, it does not generate time-lapse videos of cell cultures. To measure transfection efficiency, the cells transfected with a construct containing a reporter gene, such as gfp, are loaded into a reusable/disposable counting chamber. Next, the sample is placed on the cell counter stage and the device captures high-resolution images of the cells. The images are automatically processed using AI-based image analysis software to quantify the proportion of cells that have successfully acquired a transfected vector. The ease of use, accuracy, and reduced user-to-user variability in the assessment of cell numbers make the CytoSMART Exact FL an ideal tool for endpoint analysis of transfection efficiency.
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