A549 CELL LINE

A549 Transfection

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. Cancer research is a rapidly evolving field, and new tools and techniques are constantly being developed to improve our understanding of the disease and identify potential new treatments. One of the most versatile tools in cancer research is the A549 cell line.

A549 cells have been used in a variety of cancer research studies, including those investigating the mechanisms of cancer cell growth, survival, apoptosis, and the effects of new anticancer drugs. When cultured in vitro, these cells become a monolayer and adhere to the surface of the culture flask. These cells are also important because they make lecithin and have high levels of fatty acids, important for maintaining membrane phospholipids. The 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 Transfection Kit is available from Altogen Biosystems, which includes:

  • A549 Transfection Reagent (0.5 ml / 1.5 ml / 8.0 ml)
  • Transfection Enhancer (0.5 ml), and
  • Complex Condenser (0.5 ml)

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 described below:

  1. Plate 10,000-15,000 A549 cells per well in 0.5 ml of complete growth medium 12-24 hours before transfection
  2. Wash with 1xPBS and add 0.5 ml of fresh growth medium
  3. Prepare transfection complexes: Mix 40 µl of serum-free medium, 5.5 µl of transfection agent, and (referred to a final volume including growth medium)
    • 750 ng DNA (or mRNA), or
    • 30 nM- 50nM of siRNA (or microRNA)
  4. Incubate transfection complexes at RT for 15-30 minutes
  5. Optional: Add 2 µl of Complex Condenser. This reagent will increase transfection efficiency by reducing the size of transfection complex; however, it may increase cell toxicity
  6. Add prepared transfection complexes to 0.5 ml of complete growth medium with A549 cells (from step 2)
  7. Incubate cells at 37ºC in a humidified CO2 incubator
  8. Assay for phenotype or target gene expression 48-72 hours after transfection

Optional: Transfection efficiency can be increased by the addition of a Transfection Enhancer reagent. Add 2 µl of Transfection Enhancer reagent 12-24 hours after transfection

If the viability of A549 cells being transfected is affected at 16-24 hours post-transfection, the cytotoxicity level can be reduced by changing the growth medium and eliminating redundant exposure of cells to transfectant .

Commercially available A549 transfection reagent is provided by Altogen Biosystems – here is a link to manufacturers product page: A549 Transfection Kit

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 choose 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 is a cell line derived from human lung adenocarcinoma and in recent years this cell line have also been used in a number of cutting-edge studies utilizing cutting-edge techniques such as CRISPR-Cas9 gene editing. It is one of the most commonly used cell lines in cell biology and cancer research. Researchers use A549 cells in animal models developing new medicines that are tested in human clinical trials. Cancer is a disease that has long been considered a death sentence. But recent advances in cancer treatment are providing new hope to patients and their families. 

A549 Transfection Services

Transfection services using A549 cells (assay development, ELISA, IC-50, cell cycle, apoptosis, proliferation, A549 CDX xenograft mouse models) are offered by Altogen Labs (www.altogenlabs.com).

Publications

  • 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: https://dx.plos.org/10.1371/journal.pone.0089534

Links

Lung cancer is the major cause of cancer‑associated mortality in the world, and the invasive and metastatic characteristics of lung tumor cells are responsible for their high malignancy. Protease‑activated receptor 1 (PAR1) is a G‑protein‑coupled receptor (GPCR) activated by a special proteolytic mechanism. 

A549 CELL | A549 Cell Culture Information | A549 Transfection | Get A549 Transfection Reagent