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Original link:Metabiology - Cellular Functional Experiments
cell proliferation
Cell proliferation, apoptosis, and cell cycle are important phenotypes in tumor research, and are one of the problems solved by molecular biology and pharmacology research. By overexpressing or interfering with a certain gene in cells, studying the impact of a gene on cell proliferation ability, further studying the function of the gene, or conducting drug treatment on cells, studying the effect of drugs on proliferation.
Experimental Principles (CCK-8)
Cell proliferation is an important life characteristic of organisms, and cells proliferate through division, which is the basis for the growth, development, reproduction, and genetics of organisms. There are many research methods for cell proliferation, mainly including CCK8/CellTiter Glo ™ Wait for the method.
Cell Counting Kit-8（abbreviationCCK-8）The reagent can be used for simple and accurate analysis of cell proliferation and toxicity. The basic principle is that the reagent contains WST-8 [chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfonobenzene) -2H-tetrazole monosodium salt], which is reduced by dehydrogenase in cells to a highly water-soluble yellow formazan product (Formazan die) under the action of electron carrier 1-methoxy-5-methylphenazine sulfate dimethyl ester (1-methoxy PMS). The number of generated Jia Zan substances is directly proportional to the number of live cells. Therefore, this characteristic can be directly used for cell proliferation and toxicity analysis.
Advantages of CCK-8 method:
1. Easy to operate, avoiding the influence of human factors during the cell counting process;
2. Low cell dosage, high detection sensitivity, and stability;
3. The enzyme-linked immunosorbent assay (ELISA) reader can be used repeatedly, and the detection time is flexible, without affecting the subsequent experiments of cells;
The Formazan produced by CCK-8 is water-soluble and does not require liquid exchange, making it particularly suitable for suspended cells. Compared with MTT method, it is more convenient and safe.
Purpose: To investigate the effect of target genes on cell proliferation ability or the effect of drugs on cell proliferation ability
Materials: Target cells, stable transgenic strains (empty space, target gene)
Steps: Cell collection - Cell laying - (drug treatment) - Cell culture for 0, 6, 24, 48, and 72 hours, then add CCK-8 treatment and detect with an enzyme-linked immunosorbent assay (ELISA) reader
Experimental Principles(CellTiter-Glo™)
ATP adenosine triphosphate (ATP adenosine triphosphate) participates in various enzymatic reactions in organisms and is an indicator of live cell metabolism. Its content directly reflects the number and state of cells. During the experiment, an equal volume of CellTier Glo was added to the cell culture medium ™ Reagents measure the luminescence value. In the light signal and system, the luminescence value is directly proportional to the amount of ATP, which is positively correlated with the number of live cells. Therefore, cell viability can be obtained by detecting the ATP content.
Advantages of CTG method：
Compared with ordinary MTT and CCK8 methods, the detection reagents of the CellTiter Glo? Luminescent live cell detection system have the highest sensitivity and longer signal duration. This system has been widely used in the field of life science research, such as activity detection of some bioactive factors, large-scale screening of anti-tumor drugs, cytotoxicity tests, and tumor radiosensitivity determination;
2 CellTiter-Glo ™ The reagents are compatible with commonly used culture media in cell culture, such as RPMI1640, MEM, DMEM, and Ham's F12, and are not affected by phenol red and organic solvents, with small errors and high accuracy.
Purpose: To investigate the effect of target genes on cell proliferation ability or the effect of drugs on cell proliferation ability
Material: Target cells
Steps: Cell collection - Cell laying - (drug treatment) - Cell culture for 0, 24, 48, 72, and 96 hours, then add CellTiter Glo ™ The solution was shaken with a microplate shaker for 5 minutes and left at room temperature for 10 minutes - the fluorescence value was detected by an enzyme-linked immunosorbent assay (ELISA) reader
Cell apoptosis
Apoptosis is a focus of tumor and developmental research, as well as a hot topic in pharmacological research. Apoptosis refers to the process in which a cell, under certain physiological or pathological conditions, follows its own program and autonomously terminates its life. It is an active, highly ordered, gene controlled process involving a series of enzymes. The process of cell apoptosis can be roughly divided into the following stages: receiving apoptotic signals → regulating the interactions between apoptotic molecules → activating proteolytic enzymes (Caspases) → entering a continuous reaction process.
Basic Principles
Cell apoptosis detection was performed using the Annexin V/PI dual staining method. Annexin V is a Ca2+dependent phospholipid binding protein with a molecular weight of 35-36kD, which can bind specifically to phosphatidylserine (PS) with high affinity. In the early stages of cell apoptosis, PS can flip from the inner side of the cell membrane to the surface of the cell membrane and be exposed to the extracellular environment. Annexin V can be labeled with fluorescein (FITC or PE) or Biotin, and labeled with Annexin V as a fluorescent probe. Flow cytometry or fluorescence microscopy can be used to detect the occurrence of cell apoptosis.
7-AAD, similar to propidine iodide (PI), is a nucleic acid dye that cannot penetrate the complete cell membrane. However, in the late stage of apoptosis and dead cells, 7-AAD can penetrate the cell membrane and cause the nucleus to become red stained. Therefore, by matching Annexin V with 7-AAD, cells in the early and late stages of apoptosis can be distinguished from dead cells.
Objective: To obtain cell lines overexpressing a certain gene through lentiviral infection, and detect the apoptosis of normal cells, control group cells, and gene overexpression group cells using Annexin PE and 7-AAD double staining methods, in order to study the effect of genes on cell apoptosis.
Materials: Plasmids and cell lines
Step: Cell preparation - Collect cells - Annexin PE and 7-AAD for double staining - Flow cytometry detection
Cell invasion and migration
The ability of cells to enter the circulatory system is an important research object during the development of tumors. The related signaling pathways can mainly be divided into controlling cell adhesion and controlling the cytoskeleton. The migration and invasion of cells are mainly related to the cytoskeleton and adhesion. The changes in the invasion and migration ability of tumor cells are usually detected using Transwell. Cell scratch is also a method for measuring the motility characteristics of tumor cells, but its application is limited due to the inability to distinguish between normal proliferating and migrating cells.
Transwell experiment
A permeable membrane (usually polycarbonate membrane) at the bottom of the Transwell chamber is placed in a well plate, with the chamber referred to as the upper chamber and the culture plate referred to as the lower chamber. Due to the permeability of polycarbonate membranes, the components in the lower culture medium can affect the cells in the upper chamber. Transwell can be used to study the effects of components in the lower culture medium on cell growth, motility, and other factors.
Tumor migration experiment: studying the migration ability of tumor cells or the migration ability of tumor cells under specific circumstances. Commonly used are 8.0 and 12.0 µ m membranes. Tumor cells are seeded in the upper chamber, and FBS or certain specific chemokines are added to the lower chamber. Tumor cells will run towards the lower chamber with high nutritional content. Counting the number of cells entering the lower chamber can reflect the migration ability of tumor cells.
Tumor invasion experiment: studying the invasive ability of tumor cells or the invasive ability of tumor cells under specific circumstances. Apply a layer of matrix adhesive on the polycarbonate membrane to mimic the extracellular matrix. Tumor cells are seeded in the upper chamber, while FBS or certain specific chemokines are added to the lower chamber. Cells must digest the matrix before migrating from the upper chamber to the lower chamber. Count the number of cells entering the lower chamber to determine their invasive ability.
Purpose: To investigate the effect of overexpression/interference of target genes on cell invasion and metastasis ability
Materials: Normal cells, control lentivirus, overexpressed gene lentivirus
Steps: Cell Resuscitation - Transwell Planking - Cell Culture and Staining - Photography
Principle of Scratch Experiment
Tumor cells still have the ability to migrate in vitro. The cell scratch method is one of the methods used to determine the motility characteristics of tumor cells. It draws inspiration from the in vitro cell induced wound healing experimental model, scratches the wound on single-layer cells cultured in vitro, and then compares the migration ability of tumor cells in different experimental groups.
Purpose: Using screened stable strains (control virus, overexpressed gene virus), statistical analysis of scratch width was conducted on three groups of cells: empty cells, control stable transformation, and target gene stable transformation, to study the effect of genes on cell migration ability.
Materials: Virus and cell strains, target cells sourced from customers, stable strains and meta construction
Steps: Cell laying - Marking - Cell culture and photography - Statistical analysis
Cell cycle detection
The cell cycle refers to the entire process that a cell undergoes from the completion of one division to the end of the next division, divided into two stages: interphase and division. The cell cycle reflects the rate of cell proliferation, and the measurement of individual cell cycles can be done using time-lapse photography, but it cannot represent the cell population cycle. Therefore, other methods are often used to measure the population cycle.
principle：
The DNA content varies at different stages of the cell cycle. Typically, the G0/G1 phase of normal cells has the DNA content of diploid cells (2N), while the G2/M phase has the DNA content of tetraploid cells (4N), while the DNA content of the S phase is between diploid and tetraploid cells. PI, also known as propidium iodide, can bind to intracellular DNA and RNA. After RNA is digested by RNA enzymes, the fluorescence intensity of PI bound to DNA detected by flow cytometry directly reflects the amount of DNA in the cell.
Therefore, when using flow cytometry PI staining to detect intracellular DNA content, the cell cycle phases can be distinguished into G0/G1 phase, S phase, and G2/M phase, and the percentages of each phase can be calculated using special software.
Therefore, when using flow cytometry PI staining to detect intracellular DNA content, the cell cycle phases can be distinguished into G0/G1 phase, S phase, and G2/M phase, and the percentages of each phase can be calculated using special software.
Materials: Target cells, viruses
Steps: Cell preparation - Sample collection - Machine testing - Data analysis
Clone formation experiment
Principle: When a single cell proliferates for more than 6 generations in vitro (for about 1 week or more), the cell population formed by its offspring is called a colony or clone. Each clone contains more than 50 cells, ranging in size from 0.3 to 1.0mm. The survival rate of cell inoculation only represents the number of cells that adhere to the wall after inoculation, but not all cells that adhere to the wall may be able to proliferate and form clones. Only cells with both adherent and proliferative activity will form clones. The clone formation rate reflects the strength of a cell's independent survival ability and is used to evaluate the population of cellsDependability and proliferation ability. Due to different biological characteristics of cells, there is also a significant difference in cell clone formation rate. Generally, the clone formation rate of primary cultured cells is weak, while the passaged cell lines are strong; The formation rate of diploid cell clones is weak, while the transformed cell lines are strong; Normal cell clone formation rate is weak, while tumor cells are strong. The clone formation rate is related to the inoculation density to a certain extent. When measuring the clone formation rate, the inoculated cells must be dispersed into a single-cell suspension and directly inoculated into a culture dish for more than a week. Check at any time and terminate the culture when the cells form clones.
Objective: To study the effect of genes on cell population dependence and proliferation ability through statistical analysis of the number of clone formation in the target gene overexpression/interference cell group, empty cell group, and negative control group (empty virus).
Materials: Viruses and cell strains
Steps: Planking - Cell Culture - Observe Clones, Stain, and Calculate Clone Formation Rate
