what are the possible reason(s) that make AsPC-1, 293T and MCF-7 cell lines failed to be transfect/transduced?. Please help me with possible hints?
Hello Mansur Dabai Salisu
You may consider the following reasons.
1. The choice of which cell type to use for transfection is a critical factor that is often overlooked. Since each cell type is likely to respond differently to a given transfection reagent, choosing an appropriate cell type is necessary to maximize results. For instance, some cell types like MCF7 or HepG2, prefer to grow in clumps or clusters which is not ideal for transfection because minimal membrane surface is exposed which compromises uptake. Blood or immune cells that lack the proper endocytic machinery can minimize uptake and there are the macrophages that have an evolved uptake mechanism, but quickly breakdown and destroy endosomal contents. So, select the right cell line for transfection.
2. Make sure that the cells are not 100% confluent (should be about 60-80% confluent) or in stationary phase at the time of transfection because actively dividing cells take up foreign nucleic acid better than quiescent cells.
3. Most cell types should be used between 4 and 25 passages for optimal transfection.
4. There could be a possibility that the DNA concentration may be too low, or DNA may be degraded. In such a case, you may increase the ratio of DNA :transfection reagent, and you may confirm DNA integrity by A260/A280 spectrophotometer reading (should be at least 1.7).
5. You may use serum-free media for DNA dilution because some serum proteins may interfere with complex formation. You may also increase complexing reaction incubation time.
6. Test the culture for contamination.
7. Do not use antibiotics at the time of transfection because cationic lipid reagents increase cell permeability. As a result they may also increase the amount of antibiotics delivered into the cells, causing cytotoxicity and low transfection efficiency.
8. Cells could suffer mechanical damage during the experimental steps. So, do not vortex or spin cells for extended period.
9. Some transfection reagents could be compromised due to improper storage.
Best.
Mansur Dabai Salisu
Here are a few things that you should consider.
Transfection Reagent: Selecting the right transfection reagent is essential for achieving successful results. Certain reagents may not effectively transfect these particular cell lines.
DNA Quality and Quantity: Using transfection vector DNA of low quality or insufficient quantity can result in failed transfections.
Cell Health and Viability: Cells that are unhealthy or have low viability are less likely to successfully undergo transfection.
Transfection Conditions: Inappropriate incubation times, media conditions, or antibiotic selection can adversely affect transfection efficiency.
These hints might be a good starting point for troubleshooting
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