A549 cells are a cell line derived from an adenocarcinoma, originally developed in 1972 by removing and culturing lung tumor tissue from a 58-year-old Caucasian male patient. Since alveolar macrophages are the first cells to encounter bacterium which are inhaled or consumed, epithelial lung cells like A549, are an ideal line with which to study human infections initiated within lung tissue.
When cultured in vitro, these cells become a monolayer and adhere to the surface of the culture flask. In addition, A549 cells may be grown as a suspension. These cells are also important because they make lecithin and have high levels of fatty acids, important for maintaining membrane phospholipids. A549 cells are an important model for drug metabolism and are also used as a host for gene transfection.
A549 Cell Transfection Protocol
A pre-optimized A549 cell line transfection kit is available from Altogen Biosystems – A549 Transfection Kit. The kit is optimized to transfect siRNA, miRNA or DNA plasmid following either a standard or reverse transfection. The optimized protocol for a 24-well plate to transfect A549 cells is here:
- Prepare A549 cell suspension:
- Trypsinize cells (0.05% Trypsin) for 3-5 minutes at 37°C
- Dilute in complete growth medium to 5 x 104 cells/ml
- Prepare transfection complexes by mixing 40 µl of serum-free medium, 5.5 µL of transfection reagent, and
- 750 ng DNA (or mRNA), or
- 30 nM – 50 nM of siRNA (or microRNA)
- Incubate transfection complexes at RT for 15 – 30 minutes
- Optional: Add 2 µl of Complex Condenser. This reagent reduces the size of transfection complex, therefore increasing transfection efficiency; however it may increase cell toxicity
- Plate 20,000 – 30,000 cells per well in 0.5 mL of complete growth medium (from step #1) into culture plate
- Add prepared transfection complexes (from step 3 or 4)
- Incubate cells at 37ºC in a humidified CO2 incubator
- Assay for phenotype or target gene expression 48 – 72 hours after transfection
Scaling Transfections Up or Down
Well size of a transfection reaction depends on the intended downstream cell based assay. Moving a transfection from a 6-well plate to a 96-well plate, or vice versa, and wanting to achieve identical results is not a trivial pursuit. It is no surprise that lipoplexes stick to the plastic surfaces of cell culture dishes. As one can conclude, this significantly decreases the amount of freely available complexed material that can transfect the cells in the well. Many transfection reagent manufacturers provide a scaling factor between differing plate sizes; thus, setting up the researcher for failure if no optimization is performed. How does a researcher chose a single multiplication cofactor to determine transfection protocol scaling? Not considered is the working volume depth of media in the well. This depth varies between wells of different sizes; thus, the surface area on the inside wells in which the lipoplexes adhere to is altered. Experimental scaling is based on only growth area, well diameter or well volume, one must take into consideration the working volume. Is there enough media in each well such that the miniscus is appropriate to not cause cells to adhere to only the outer rim of the well? Maintaining concentration based on final working volume can help maintain oligonucleotide and transfection reagent concentrations, but cell number confluency will need to be tested due to inconsistencies of ratios between working volume and growth area. These reasons are why optimized parameters determined for one plate format cannot be used as a set ratio when scaling experiments up or down. Changing a plate format requires conditions to be re-optimized for the new plate size.
Table: Cell culture plate working volumes and dimensions. The ratio was determined as the ratio of the 6-well value to the 96-well value. The result is that a single parameter cannot be used for transfection scaling purposes.
A549 Transfection Services
- Use of A549 cells as a model for human lung epithelium: This study used a range of stains and tracking molecules to examine the trafficking of molecules through the cell. Experimental results agreed well with literature values for primary culture of Type II pulmonary epithelium, indicating that A549 cells are a useful model for studying these types of cells in regards to metabolism and processing of macromolecules. LINK: http://www.ncbi.nlm.nih.gov/pubmed/9743595
- Exosome function in the presence of cisplatin in A549 cells: Exosomes are utilized by cells for communication and removal of cellular waste. In the presence of cisplatin (a chemotherapeutic drug), this study found A549 cells increased their exosome secretion. Subsequently, neighboring A549 cells developed an increased resistance to cisplatin. The study theorizes that inhibition of exosomes may be a novel future treatment for lung cancer. LINK: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0089534