Cell Experiments

• Q1: What are the factors affecting cell transfection experiments?

  A：

  1) Transfection reagents The transfection efficiency of different cell lines is usually different, but the selection of cell lines is usually based on the needs of the experiment. Therefore, before the transfection experiment, a suitable transfection reagent should be selected based on the experimental requirements and cell characteristics. Each transfection reagent will provide a list of cell lines and literature that have been successfully transfected. Through this information, you can choose the transfection reagent that best suits the experimental design.

  2) The cell growth status of generally low cell passages (<50) can ensure that the genotype remains unchanged. The most suitable cells for transfection are cells that have reached the exponential growth phase after several passages. The cells grow vigorously and are easiest to transfect. Cell cultures can evolve through mutations, gross chromosomal rearrangements, or changes in gene regulation after being stored in the laboratory for months and years. This can lead to changes in cell behavior relevant to transfection. That is to say, the biological properties of cell lines of the same strain will change to varying degrees under different culture conditions in various laboratories, resulting in changes in their transfection characteristics. Therefore, if you notice a decrease in transfection efficiency, you can try transfecting freshly cultured cells to restore optimal results.

  3) Transfection methods may have different transfection effects due to differences in the nature of cells cultured in different laboratories, differences in plasmid quantification, differences in operating techniques, etc. The optimal transfection conditions should be determined according to the specific conditions of the laboratory.

  4) Vector structure The construction of the transfection vector (viral vector, plasmid DNA, RNA, PCR product, oligonucleotide, etc.) also affects the transfection results. Viral vectors are more efficient at infecting specific host cells, but different viral vectors have specific hosts, and some require specific cell cycles. For example, retroviruses need to infect host cells in the dividing phase. In addition, some safety issues need to be considered. (such as genetic contamination). In addition to vector construction, the shape and size of the vector also have different effects on transfection efficiency, such as the aforementioned effects of supercoiled and linear DNA on transient and stable transfection. If the gene product has a toxic effect on cells, transfection will be difficult, so it is also important to select a promoter with a composition that can be regulated or with appropriate strength. At the same time, a transfection positive control constructed with the same vector of the empty vector and other genes can eliminate toxicity. influence interference.

• Q2: In virus-infected cell experiments, what is the reason for cytotoxicity when using infection enhancers?

  A：

  1) If the dosage of enhancer is too large, its dosage can be appropriately reduced;

  2) If the amount of virus is too large, the amount of virus can be appropriately reduced, and the method of changing the culture medium or increasing the amount of culture medium can be used.

• Q3: What is the appropriate amount of cell inoculation for viral infection?

  A：

  1) Determine the inoculation amount based on the cell proliferation rate. Generally, it is necessary to ensure that the cells almost cover the bottom of the culture dish about 4 days after infection.

  2) For most cell lines: the passage period is 2-3 days, and the cell plating density during infection is maintained within the range of 20%-30%.

  3) For some primary cells: cells grow slowly, and the confluency can be increased to 50%-60% during inoculation, ensuring that the cell confluence reaches 90%-100% about 4 days after infection.

  4) For non-dividing cells: they no longer proliferate after inoculation (such as neuronal cells), inoculate at 100% confluence.

• Q4: After adding the virus, the cells died seriously. What is the reason? How to deal with it?
  A: Generally, viruses have certain toxicity to cells, and the infection MOI can be adjusted and lowered. And pay close attention to the cell status, and observe the cell status regularly at 4, 8, and 12 hours after infection. If the cell status deteriorates, immediately change the medium of the cells and replace the virus-infected culture medium with fresh complete culture medium.
• Q5: How to improve virus infection efficiency?
  A: The efficiency of virus infection is affected by many factors, including the growth status of the cells themselves, the number of cells, and the ease of infection of the cells. Therefore, during the experiment, ensuring normal cell proliferation and clear outlines while maintaining appropriate cell density and selecting optimal infection conditions can better ensure infection efficiency. For suspension cells, centrifugal infection can be used to reduce the volume of virus infection and thereby improve the infection efficiency. If the culture plate is sealed, centrifuge it with a square-angle rotor centrifuge at 1000 g for 1 hr, and then put it back into the incubator to continue normal culture.
• Q6: Cells can be infected by viruses, but why is GFP fluorescence so weak?
  A: After the GFP virus infects cells, the fluorescence intensity of the cells depends on the number of virus particles entering the cells, the proliferation status of the cells themselves, cell type, observation time, GFP gene promoter activity and other factors. Therefore, the more viral particles the target cells are infected with and the faster the cells proliferate, the stronger the GFP fluorescence will be. Secondly, GFP protein expression reaches its peak only 96-120 hrs after virus infection in cells that proliferate quickly; in cells that proliferate slowly, this time is longer. In addition, the fluorescent expression of the GFP gene behind a strong promoter is strong, while conversely, the fluorescent expression behind a weak promoter is weak.
• Q7: The electroporation transfection effect is not good, what are the possible influencing factors?

  A: Electroporation is an efficient and simple gene transfer system. It has advantages that other transfer methods cannot match, such as simple and fast operation, strong reproducibility, high transfection rate, and wide-spectrum applicability, especially for genes that are currently generally considered difficult to transfect. Suspension cultured cells can also achieve higher transfection. If the electroconversion effect is not good, you can consider the following conditions for optimization:

  1) Electric field parameters Electric field strength is the main parameter that should be optimized. The electric field intensity cannot be too high, as too high will increase cell death rate; nor can it be too low, because too low will not increase the permeability of the membrane or form pores on the membrane. Different cell lines have different optimal field strength values. In addition to directly measuring and comparing the transfection rates under different field strengths, the method for determining them can also adopt a simpler indirect method. Literature shows that electric field parameters with a survival rate of about 50% are ideal parameters, so the survival rate can be indirectly measured to determine the optimal field strength value.

  2) Pulse process
  ① Pulse waveforms are mainly divided into two types, square wave pulse and exponentially decreasing wave pulse. Generally, mammalian cells are electroporated with square wave pulses, while bacteria, yeast, and insect cells are electroporated with exponentially decreasing wave pulses.
  ②The selection of pulse time mainly depends on the pulse waveform. In square wave pulses, the pulse time can be set directly. In exponentially decreasing wave pulses, the pulse time refers to the time it takes for the voltage to decay to 1/3 of the initial voltage, which is equal to the product of capacitance (C) and resistance (R), and the unit is ms. In parameter optimization, increasing the voltage should decrease the pulse time, while decreasing the voltage should increase the pulse time.
  ③In general, single pulse is chosen for most cell types. In some cases, multiple pulses may be used, because low voltage, short pulse time, and multiple pulses can effectively avoid cell damage. It is recommended that the interval between multiple pulses is 1 minute.

  3) Cell factors Cells used for electroporation are generally selected to be in the logarithmic growth phase (within 15 generations, 2 days after passage). The cell suspension concentration is generally 1*106/ml. When the cell growth density is greater than 3*106/ml, the transfection efficiency will decrease.

  4) Plasmid factors From the perspective of plasmid concentration, the cell density is 1*106/ml, and the DNA dosage is 2-5 μg/ml for the highest transfection efficiency.

  5) Temperature Under normal circumstances, the electroporation process is performed at room temperature, but the cells need to be treated in an ice bath before and after electroporation.

  6) Selection of culture medium after electroporation. Cells are very fragile after electroporation. The choice of culture medium should focus on improving the survival rate of cells. Generally, hypotonic RPMI 1640+10% FCS is selected. In addition, you can refer to adding 50 mmol/L trehalose + 1.25% DMSO.

• Q8: What are the methods for introducing siRNA into cells, and which one is best?
  A: There are several methods to introduce siRNA into cells: chemical transfection technology, electroporation method, calcium phosphate co-precipitation technology, microinjection and vector introduction technology. When selecting, the following factors should be considered based on the experimental conditions: the ability of the cells to withstand the transfer method, the susceptibility of the cells to virus infection, the growth characteristics of the cells, etc. Chemical transfection technology is the most commonly used method for adherent cells, while electroporation is more effective for suspension cells.
• Q9: How to detect the effect of transfection in vivo?
  A: It is generally carried out according to the research requirements of the project. If you simply test the transfection efficiency, it is recommended to use the qRT-PCR method and the luciferase method. It can also be done using histological and physiological methods, as well as physical measurements, depending on the research. Due to the complexity of in vivo transfection, the method of transfecting GFP protein and observing it with a fluorescence microscope is usually used in in vitro experiments. Due to the high background of tissue materials taken out from the body, detection is difficult.
• Q10: What are the factors that affect siRNA transfection efficiency?

  A：

  1) The ratio of RNA to transfection reagent is not good. Due to differences in RNA sequences, different synthesis conditions, and whether it is labeled with fluorescence or other markers, RNA and transfection reagents will have different optimal conditions under different circumstances. It is recommended to conduct preliminary experiments first. optimization.

  2) The cell density is not good. Adjust the cell density to a confluency of 20-40% during transfection. Cells that are successfully transfected with siRNA will have down-regulation of target gene expression, but cells that are not successfully transfected will not be affected. At this time, the transfection efficiency and the total number of cells are very important. Generally, the transfection efficiency is high when the number of cells is small. Due to the timeliness of siRNA silencing, qRT-PCR detection can only be performed 48 hours after transfection, and protein detection can only be performed 48-72 hours after transfection. If the plating density is high during transfection, on the one hand, the transfection effect of the cells will not be ideal, which directly affects the silencing effect and data reliability. On the other hand, after 48 hours or even longer, when the optimal point of silencing detection is reached, the cells will be too dense. will affect the cell state and thus the experimental results.

  3) The RNA efficiency is not high. Select the optimal RNA and use known efficient RNA as a control. When synthesizing siRNA, attention must be paid to selecting high-purity siRNA. The purity of siRNA is directly related to transfection efficiency and silencing efficiency.