Question

How many passages should the cell line go through before the transfection experiment to assure that the cells in the culture plate exhibit high viability and are in the log phase of growth?

Answer 1

The various passages that a cell line should go through before tranfection are as

1.cell type :-The choice of which cell type to use for a transfection experiment may seem obvious, but it is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent or method, choosing the appropriate cell type and proper experimental design are necessary to maximize results.

2.cell health and viability:-The viability and general health of cells prior to transfection is known to be an important source of variability from one transfection to another. In general, cells should be at least 90% viable prior to transfection and have had sufficient time to recover from passaging

3.Confluency:-For optimal transfection results, follow a routine subculturing procedure and passage cultures once or twice a week at a dilution that allows them to become nearly confluent before the next passage. Do not allow the cells to remain confluent for more than 24 hours

4.Media:-Different cells or cell types have very specific medium, serum, and supplement requirements, and choosing the most suitable medium for the cell type and transfection method plays a very important role in transfection experiments.

5.Serum:-In general, the presence of serum in culture medium enhances transfection with DNA. However, when performing cationic lipid-mediated transfection, it is important to form DNA-lipid complexes in the absence of serum because some serum proteins interfere with complex formation

6.Antibiotics:-

antibiotics can be present in the medium for transient transfection. However, because cationic lipid reagents increase cell permeability, they may also increase the amount of antibiotics delivered into the cells, resulting in cytotoxicity and lower transfection efficiency. Therefore, we do not recommend adding antibiotics to the transfection medium. Avoiding antibiotics when plating cells for transfection also reduces the need for rinsing the cells before transfection.

7.Type of molecule transfected:-Plasmid DNA is the most commonly used vector for transfection. The topology (linear or supercoiled) and the size of the plasmid DNA vector influence the efficiency of transfection. Transient transfection is most efficient with supercoiled plasmid DNA.

8.Transfection method:-There are a number of strategies for introducing nucleic acids into cells that use various biological, chemical, and physical methods. However, not all of these methods can be applied to all types of cells and experimental applications, and there is a wide variation with respect to transfection efficiency, cell toxicity, effects on normal physiology, level of gene expression etc.