Mastering Fluorochrome-labeled Cells: From Principles to Engineering

In the microscopic world of life sciences, fluorochrome labeling has become a powerful technique for visualizing cells with high sensitivity and specificity—without disrupting their normal physiological functions. It is increasingly essential for exploring cellular mechanisms and has led to major breakthroughs in life science research. In this issue of the Cell Culture Academy, we’ll dive into the fluorochrome-lit world of cells together.

Ⅰ. Definition, Application, and Principles of Fluorochrome-labeled Cells

1. Definition

Fluorochrome-labeled Cells are created by introducing fluorochrome proteins or luciferase reporter genes into host cells using transgenic techniques. This allows real-time visualization of cell states, behaviors, and molecular processes. Common fluorochrome proteins include green fluorescent protein (GFP) and red fluorescent protein (RFP), among others.

2. Application

Fluorochrome-labeled cells are extensively used across a variety of fields, including basic research and drug development. Common applications are summarized in Table 1.

Table 1. Applications of Fuorochrome-labeled Cells

3. Principles

Fluorochrome-labeled cells are typically generated using lentiviral vectors. The process involves several key steps:

A. A lentiviral vector is engineered to carry a fluorochrome protein gene (e.g., GFP or mCherry) or a reporter gene (e.g., luciferase). The vector is then packaged into infectious lentiviral particles. B. These particles utilize the VSV-G envelope protein to bind to phospholipid components of the host cell membrane, facilitating viral entry via endocytosis. Once inside the host cell, the viral RNA genome is released and reverse transcribed into proviral DNA by reverse transcriptase. This proviral DNA is then randomly integrated into the host genome with the help of integrase, enabling stable expression of the fluorochrome protein gene.

C. Cells expressing the fluorochrome protein are selected using antibiotic resistance (e.g., puromycin) or isolated by flow cytometry, resulting in the establishment of a stable fluorochrome cells.

Ⅱ. Advantages and Limitations of Lentiviral Vectors

Advantages

A. Broad host range: Lentiviruses can efficiently infect both dividing and non-dividing cells.

B. High transduction efficiency in hard-to-transfect cells: They are particularly effective at delivering genes to challenging cell types, such as primary cells, stem cells, and undifferentiated cells, as well as cells that mount strong immune responses to adenoviruses (e.g., dendritic cells and mesenchymal stem cells).

C. Low immunogenicity: Lentiviral vectors induce minimal immune responses, even when directly injected into living tissues, making them well-suited for in vivo animal studies.

D. Cell-type specificity: Tissue-or cell-specific promoters and enhancers (e.g., neuronal promoters) can be incorporated into lentiviral constructs to achieve targeted gene expression in specific cell populations.

Limitations

Despite their numerous advantages, lentiviral vectors do have some limitations, particularly regarding biosafety and viral titer. Most commonly used lentiviral systems are derived from HIV-1 and are based on a three-or four-plasmid packaging system. These systems remove packaging signals and introduce exogenous target genes to produce pseudotyped viruses that cannot replicate or produce new virions in host cells. However, since lentiviruses naturally infect humans, biosafety concerns remain, especially in clinical or large-scale applications. As a result, ongoing efforts focus on optimizing vector design and increasing viral titers to enhance the safety and broaden the application of lentiviral vectors in research and therapy.

III. Simplified Protocol for Engineering GFP-labeled Cells

1. Cell Line Selection and Condition Assessment

Lentiviral Packaging Cells: Use HEK-293T cells for lentivirus production. Ensure the cells are in the logarithmic growth phase and in good condition.

Target Cells: Select the desired cell line (e.g., A549, HepG2). Ensure the cells are in the logarithmic growth phase and in good condition.

2. Lentivirus Production
Co-transfect HEK-293T cells with the lentiviral packaging system using a liposome-based transfection reagent. This system typically includes the transfer plasmid (containing GFP and puromycin genes), the packaging plasmid, and the envelope plasmid.

3. Lentiviral Infection

A. MOI Optimization: Determine the optimal multiplicity of infection (MOI) through preliminary experiments to achieve a balance between transduction efficiency and cytotoxicity.

B. Cell Seeding: Digest and collect healthy target cells, resuspend them, and seed into culture plates at an appropriate density (typically 1×105 cells/mL). Incubate overnight at 37℃ with 5% CO₂.

C. Infection Procedure: Thaw the lentiviral stock on ice. Replace the medium with fresh medium, then add the virus at the optimized MOI along with polybrene (commonly at 8 μg/mL) to enhance infection. Incubate overnight.

4. Antibiotic Selection

A.Puromycin Titration: Perform a preliminary test with a range of puromycin concentrations to determine the minimum lethal dose for the specific cell line.

B.Selection: At 48 h post-infection, replace the medium with complete medium containing puromycin at the predetermined concentration. Refresh the selection medium every 2-3 d and continue culturing until only stable, GFP-positive cells remain.

5. Cell Expansion
A. Once stable GFP-expressing cells are established, reduce the puromycin concentration by half for long-term culture.

B.Refresh the selective medium every 2-3 d to maintain stable expression.

6. Validation and Cryopreservation
A. Analyze GFP expression using flow cytometry to confirm transduction efficiency.

B. Once the desired positivity threshold is reached, switch to regular complete medium.

C. Cryopreserve the cells for future experiments.

IV. Case Sharing

Following the protocol described above, the lentiviral packaging plasmids were transfected into HEK-293T cells, resulting in the successful production of lentiviral particles carrying the GFP gene (Fig. 1).

These viral particles were used to infect A549 and HepG2 cells. Prior to infection, puromycin sensitivity curves were established for both A549 and HepG2 cells. Cells were exposed to a range of puromycin concentrations (0, 0.5, 1.0, 1.5, and 2.0 μg/mL) to identify the minimum lethal concentration. Based on the results, 1.5 μg/mL was determined to be the appropriate selection concentration for both cell lines (Fig. 2).

Using this optimized concentration, stable GFP-positive clones were successfully selected and expanded, confirming the effective construction of fluorochrome-labeled cells (Fig. 3).

Fig. 1. GFP Expression following Lentiviral Plasmid Transfection in HEK-293T Cells (Left: 40×; Right: 100×)

	A549	HepG2
0 μg/mL
0.5 μg/mL
1.0 μg/mL
1.5 μg/mL
2.0 μg/mL

Fig. 2. Cell Morphology of A549 and HepG2 Cells after 72 h of Puromycin Treatment (100×)

(A)  Puromycin 0 μg/mL: Nearly all cells survive.

(B)  Puromycin 0.5 μg/mL: Minor cell death.

(C)  Puromycin 1 μg/mL: Few cells remain.

(D)  Puromycin 1.5 μg/mL: Nearly all cells die.

(E)  Puromycin 2 μg/mL: Complete cell death.

A549
HepG2

Fig. 3. GFP Expression in A549 and HepG2 Cells after Puromycin Selection (Left: Fluorescence Images (100×); Right: Flow Cytometry Analysis)

V. Precautions

1. Plasmid quality for virus packaging: The purity and concentration of nucleic acids greatly affect transfection efficiency. It’s recommended to use plasmids with a concentration of 400-1,000 ng/μL and an A260/280 ratio between 1.8-2.0.

2. Virus handling: Avoid repeated freeze-thaw cycles, as they can significantly reduce viral titer. To preserve activity, aliquot the virus based on expected usage. If the virus has been stored for more than 6 months, recheck the titer before use.

3. Hard-to-infect cells: For cell types that are difficult to infect (e.g., dendritic cells), consider multiple rounds of infection. A second infection with fresh virus 24 hours after the first can greatly improve efficiency.

4. Stable cell lines: For stably transduced lines, assess fluorochrome protein expression by flow cytometry every 5-10 passages. The testing frequency may be adjusted based on experimental needs and labeling strategies. To maintain signal stability, apply selective pressure (e.g., G418 or puromycin), optimize culture conditions, and regularly cryopreserve the cells.