Original article link: HeYuan Bio – Cellular Functionality Experiments

1. Cell proliferation

        Cell proliferation, apoptosis, and the cell cycle are key phenotypes in cancer research and represent some of the central questions addressed in molecular biology and pharmacology. By overexpressing or knocking down a specific gene in cells, researchers can investigate its impact on cellular proliferative capacity, thereby elucidating its functional roles; alternatively, cells can be treated with drugs to assess how these agents modulate proliferation.

Experimental Principle (CCK-8)

Cell proliferation is an essential life characteristic of living organisms. Cells proliferate through division, which underpins growth, development, reproduction, and heredity. Numerous methods are available for studying cell proliferation, with CCK‑8 and CellTiter‑Glo™ being among the most commonly used.

The Cell Counting Kit‑8 (CCK‑8) reagent enables simple and accurate assays of cell proliferation and cytotoxicity. Its underlying principle is that the reagent contains WST‑8 [chemical name: 2-(2‑methoxy‑4‑nitrophenyl)-3-(4‑nitrophenyl)-5-(2,4‑disulfophenyl)-2H‑tetrazolium monosodium salt], which, in the presence of the electron‑coupling agent 1‑methoxy‑5‑methylphenazinium methyl sulfate (1‑Methoxy PMS), is reduced by cellular dehydrogenases to a highly water‑soluble yellow formazan dye. The amount of formazan produced is directly proportional to the number of viable cells; thus, this property can be exploited for direct quantification of cell proliferation and cytotoxicity.

Advantages of the CCK-8 method:

　　 1. Simple operation, minimizing the influence of human error during cell counting;

　　 2. Requires a small cell input, exhibits high detection sensitivity, and is stable;

　　 3. The plate can be repeatedly read using a microplate reader, allowing flexible assay timing without interfering with subsequent cell-based experiments.

　　 4. The formazan produced by the CCK‑8 assay is water‑soluble, eliminating the need for medium replacement; it is particularly well suited for suspension cells and is more convenient and safer than the MTT assay.

　　Objective: To examine the effects of the target gene on cellular proliferation or the effects of a drug on cellular proliferation.

　　Materials: Target cells, stable transfectants (empty vector, target gene)

　　Steps: Cell collection — cell plating — (drug treatment) — after culturing for 0, 6, 24, 48, and 72 hours, add CCK‑8 reagent, followed by measurement using a microplate reader.

Experimental Principle (CellTiter-Glo™)

　　ATP, or adenosine triphosphate, participates in numerous enzymatic reactions within living organisms and serves as an indicator of cellular metabolism. Its concentration directly reflects both the number of viable cells and their physiological state. During the assay, an equal volume of CellTiter‑Glo™ reagent is added to the cell culture medium, and the luminescence signal is measured. In this system, the luminescence intensity is proportional to the ATP content, which in turn correlates positively with the number of viable cells. Consequently, cell viability can be assessed by quantifying ATP levels.

　　Advantages of the CTG method:

　　1 Compared with conventional MTT and CCK‑8 assays, the CellTiter‑Glo™ luminescent live‑cell assay reagent offers superior sensitivity and a longer signal duration. This system has been widely adopted in life sciences research, including the assessment of bioactive factor activity, high‑throughput screening of anti‑tumor drugs, cytotoxicity testing, and the evaluation of tumor radiosensitivity, among other applications.

　　2 CellTiter-Glo™ reagent is compatible with commonly used cell culture media, such as RPMI 1640, MEM, DMEM, and Ham’s F12, and is unaffected by phenol red or organic solvents, resulting in low variability and high accuracy.

　　Objective: To examine the effects of the target gene on cellular proliferation or the effects of a drug on cellular proliferation.

　　Materials: Target cells

　　Procedure: Cell collection — cell plating — (drug treatment) — after 0, 24, 48, 72, and 96 hours of cell culture, add CellTiter‑Glo™ reagent, shake on a microplate shaker for 5 minutes, and incubate at room temperature for 10 minutes; then measure the luminescence using a plate reader.

1. Apoptosis

　　Apoptosis is a central focus in tumor and developmental research and also a major area of pharmacological investigation. It refers to the process by which cells, under specific physiological or pathological conditions, autonomously terminate their life in accordance with an intrinsic program. This is an active, highly orchestrated, genetically regulated process involving a cascade of enzymatic reactions. The apoptotic pathway can be broadly divided into the following stages: reception of apoptotic signals, interactions among apoptotic regulatory molecules, activation of proteolytic enzymes (caspases), and initiation of a sequential cascade of events.

　　Basic Principle

　　Apoptosis detection was performed using the Annexin V/PI double-staining assay. Annexin V is a Ca²⁺‑dependent phospholipid‑binding protein with a molecular mass of 35–36 kDa, which binds with high affinity and specificity to phosphatidylserine (PS). During the early stages of apoptosis, PS translocates from the inner leaflet of the cell membrane to the outer surface, becoming exposed to the extracellular environment. Annexin V is labeled with a fluorescent dye (FITC or PE) or biotin; the labeled Annexin V serves as a fluorescent probe, enabling the detection of apoptotic cells by flow cytometry or fluorescence microscopy.

　　7-AAD, similar to propidium iodide (PI), is a nucleic acid dye that cannot penetrate intact cell membranes; however, in cells at the late stages of apoptosis and in dead cells, 7-AAD can cross the membrane and stain the nuclei red. Therefore, by using Annexin V in combination with 7-AAD, it is possible to distinguish between early- and late-stage apoptotic cells as well as dead cells.

　　Objective: To generate cell lines with overexpression of a specific gene via lentiviral infection, and to assess apoptosis in normal cells, control cells, and gene-overexpressing cells using Annexin‑PE/7‑AAD double staining, thereby investigating the gene’s effect on cellular apoptosis.

　　Materials: Plasmids and Cell Lines

　　Steps: Cell preparation — cell collection — double staining with Annexin‑PE and 7‑AAD — flow cytometry analysis

1. Cell invasion and migration

　　During tumor progression, the ability of cells to enter the circulatory system is a key area of investigation. The relevant signaling pathways can be broadly categorized into those that regulate cell adhesion and those that govern the cytoskeleton. Cell migration and invasion are primarily driven by cytoskeletal dynamics and adhesive interactions. Changes in the invasive and migratory capacities of tumor cells are typically assessed using the Transwell assay. Scratch assays also serve as a method for evaluating tumor cell motility; however, their utility is limited because they cannot distinguish between cells undergoing normal proliferation and those engaged in migration.

Transwell assay

　　A permeable membrane (typically a polycarbonate membrane) is placed at the bottom of a Transwell insert, which is then positioned in a well plate. The chamber above the membrane is referred to as the upper chamber, while the chamber below it is called the lower chamber. Because the polycarbonate membrane is permeable, components in the culture medium of the lower chamber can influence the cells in the upper chamber. Using a Transwell system, one can investigate how factors in the lower chamber’s culture medium affect cell growth, migration, and other cellular processes.

　　Tumor migration assay: This assay evaluates the migratory capacity of tumor cells, or their migratory behavior under specific conditions. A commonly used membrane pore size is 8.0 or 12.0 µm. Tumor cells are seeded in the upper chamber, while FBS or specific chemokines are added to the lower chamber. Tumor cells migrate toward the nutrient‑rich lower compartment; the number of cells that transmigrate into the lower chamber serves as an indicator of the tumor cells’ migratory ability.

　　Tumor invasion assay: This assay evaluates the invasive capacity of tumor cells, or their invasive behavior under specific conditions. A layer of Matrigel is coated onto a polycarbonate membrane to mimic the extracellular matrix. Tumor cells are seeded in the upper chamber, while FBS or specific chemokines are added to the lower chamber. Cells must degrade the Matrigel before they can migrate from the upper to the lower chamber; the number of cells that have migrated into the lower chamber is then counted to quantify the cells’ invasive potential.

　　Objective: To investigate the effects of overexpression or knockdown of the target gene on cellular invasion and metastatic potential.

　　Materials: Normal cells, control lentivirus, and gene-overexpression lentivirus.

　　Steps: Cell resuscitation — Transwell plating — Cell culture and staining — Imaging

　　Principle of the scratch assay

　　Tumor cells retain the ability to migrate in vitro. The scratch assay is one of the methods used to assess the migratory properties of tumor cells. Drawing on an in vitro cell‑wounding and healing model, a monolayer of cultured cells is scratched to create an injury, after which the migratory capacity of tumor cells is compared across different experimental groups.

　　Objective: Using selected stable cell lines (transduced with a control virus or an overexpression‑driven virus), we performed statistical analysis of scratch‑wound widths in three groups—mock‑transduced cells, control‑stable transfectants, and target‑gene‑stable transfectants—to investigate the impact of the gene on cellular migratory capacity.

　　Materials: virus and cell lines; the target cells are provided by the customer; stable cell lines and vector constructs.

　　Steps: Cell seeding — scratch (wound-healing assay) — cell culture and imaging — statistical analysis

1. Cell cycle analysis

　　The cell cycle refers to the entire process a cell undergoes from the completion of one division to the end of the next, and it is divided into two phases: interphase and mitotic phase. The cell cycle reflects the rate of cellular proliferation; the duration of the cycle for an individual cell can be determined using time-lapse photography, but this method does not capture the behavior of a cell population. Therefore, other approaches are now commonly employed to measure the population‑level cell cycle.

　　Principle:

　　The DNA content varies across the different phases of the cell cycle: typically, normal cells in the G0/G1 phase exhibit a diploid DNA content (2N), whereas those in the G2/M phase display a tetraploid DNA content (4N), and the DNA content during the S phase falls between diploid and tetraploid levels. Propidium iodide (PI) binds to both DNA and RNA within cells; after digesting RNA with RNase, the fluorescence intensity of PI bound to DNA—measured by flow cytometry—directly reflects the cellular DNA content.

　　Therefore, when assessing intracellular DNA content by flow cytometry using propidium iodide staining, the cell cycle can be divided into the G0/G1 phase, the S phase, and the G2/M phase, and the percentage of each phase can be calculated using specialized software.

　　Objective: To generate cell lines with gene overexpression via lentiviral infection, and to assess changes in the cell cycle of each group using propidium iodide staining combined with flow cytometry, thereby investigating the impact of the target gene on the cell cycle.

　　Materials: target cells, virus

　　Steps: Cell preparation — Sample collection — Instrumental analysis — Data analysis

1. Clonogenic assay

　　Principle: When a single cell is cultured in vitro for more than six passages (typically over one week), the resulting population of descendant cells is referred to as a colony or a clone. Each clone contains at least 50 cells and measures between 0.3 and 1.0 mm in diameter. The cell seeding survival rate indicates only the number of cells that adhere to the culture surface after plating; however, not all adherent cells necessarily proliferate to form colonies. Only those cells that both adhere and exhibit proliferative capacity can give rise to colonies. The colony‑forming efficiency reflects the degree of a cell population’s ability to survive independently and is used to assess its dependence on anchorage and its proliferative potential. Due to differences in cellular biological characteristics, colony‑forming efficiencies vary considerably: primary cultures generally show low efficiency, whereas established cell lines exhibit higher efficiency; diploid cells typically display lower efficiency, while transformed cell lines demonstrate higher efficiency; normal cells tend to have lower efficiency, whereas tumor cells show much higher efficiency. Colony‑forming efficiency is also influenced by seeding density; therefore, when determining this parameter, cells should be dispersed into a single‑cell suspension and directly plated in culture dishes. Cultures are maintained for at least one week, with regular monitoring, and are terminated once colonies have formed.

　　Objective: To investigate the effects of gene expression on cellular population dependence and proliferative capacity by performing statistical analyses of colony-forming unit counts across groups comprising cells with overexpression or knockdown of the target gene, control cells, and a negative control group (empty vector).

　　Materials: Virus and cell lines

　　Steps: Plate the cells—culture the cells—observe the clones, stain them, and calculate the clone‑forming efficiency.