Transfection is a common, yet sophisticated method that is frequently used to artificially deliver nucleic acids (DNA or RNA) into cells for a variety of applications. To efficiently introduce nucleic acids to the cell, a chemical method such as lipid based reagents or a physical method such as electroporation is most commonly used. These nucleic acids can alter properties of the cell, allowing for the study of gene function and protein expression within the context of the cell. However, there are a number of important factors, such as reagent dose, nucleic acid dose, cell density, complexation media, incubation time, etc., that can affect the efficiency of transfection. The difference between a good and bad transfection, can ultimately determine how many times an experiment will be repeated. Understanding the interaction between these key factors and the importance of optimization for a particular cell type can help to reduce the cost and consumption of time and reagents.
Hello, I’m working with multiple cancer cell lines. Can I use the same amount of any transfection reagent for different cells lines?
0 0 0 Total: 0 Great question. The answer is no. Transfection efficiency depends highly on the amount of reagent used per well and maybe very different between reagents. Usually, the protocol that is supplied with the product will provide an optimal range of transfection reagent to use. You can go to www.lifetechnologies.com/us/en/home/life-science/cell-culture.html to get … Continued
Hello, I recently started a new project that requires transfection for a few cancer cell lines. I wonder if you could suggest some of the key considerations for successful transfection.
0 0 0 Total: 0 That’s a great question! Successful transfection is influenced by many factors—the choice of the transfection method, viability of the cells prior to transfection, number of passages, degree of confluency, quality and quantity of the nucleic acid used, the amount of reagent can all play a part in the outcome of … Continued
Can you describe the ideal cell culture conditions for the best transfection results and does serum in the media have any impact on transfection success?
0 0 0 Total: 0 The ideal cells are between passages 5-20 after thawing and growing in log phase. Prior to transfection, cell viability should be greater than 90% and 70~80% confluent. For transfection, we recommend using Opti-MEM (serum-free) for lipid/payload complex formation and culture medium supplemented with 10% serum. Lower amount of serum (<10%) … Continued
0 0 0 Total: 0 The best time to perform transfection after thawing depends on the cell type. A wide variety of immortalized cell lines have optimal performance after passage 4-5 and until passage 20-25. If transfection is performed on low passage cells, post thawing, lower transfection performance will be observed; while cells that are … Continued
How can you determine if your cell line will be easy or challenging to transfect? Is there anything you can do to make it easier if you have a difficult line?
0 0 0 Total: 0 Typically, we identify a cell line as “difficult to transfect” by using a positive control reporter, such as GFP or luciferase. Multiple delivery methods should also be tested for any new cell type and the most commonly and easy to use method is lipid mediated; however, if this approach doesn’t … Continued
0 0 0 Total: 0 Yes, antibiotics can be used during transfection with any of the Life Technologies lipid based reagents. However, the critical steps of the protocol need to be performed with the recommended OptiMEM Media and not culture media that contains any serum or antibiotics. The transfection protocol involves a two tube protocol … Continued
0 0 0 Total: 0 For delivery in primary cells or any cell model, gene expression is dependent on the downstream assay required for the experiment. A typical window or range is anywhere from as early as 12 hours for expression from mRNA delivery, 24-48 hours for evaluation of expression of a fluorescent protein, to … Continued
I am trying to co-transfect DNA and siRNA into HEK cells. Can you give me some advice on how the most effective way to do this.
0 0 0 Total: 0 For getting successful co-transfection, you need high quality DNA, a validated siRNA against the gene of interest and log phase growing HEK293 cells. The method or application for co-transfection is important to help you determine the order of delivery and the right reagent for transfection: 1. To study the effect … Continued
How do I address the incredible difficulty of transfecting hematopoetic cells, specifically immune system cells (e.g., macrophages such as J774A.1 and THP-1)?
0 0 0 Total: 0 Do you really need to use this cell type? These notoriously difficult cells require a certain amount preparation, because a variety of delivery techniques might need to be tested for success. If you are trying to over express a gene with DNA delivery, the newly developed Lipofectamine® 3000 can be … Continued
After transfection, I regularly get a drop in cell viability. Is there any reason why this continues to happen and what things should I be troubleshooting?
0 0 0 Total: 0 You might try removing the media that contains the transfection complex 4-6 hours post transfection and replace it with growing culture media. If this doesn’t help with cell viability, you might need to do a small experiment to optimize a few key parameters that can influence the performance of your … Continued