A549 cells are cultured in complete media consisting of Dulbecco’s MEM modified with 10% FBS.  The cells can be grown as adherent or in suspension in vitro.  The A549 cell line grows easily and cell count doubling time is typically 24-40 hours.

A549 Cell Culturing Protocol

  1. Rinse cells with 0.25% Trypsin/0.53 mM EDTA
  2. To a T75 flask, add 2 mL of trypsin-EDTA to the flask and observe for cell layer detachment under an inverted microscope.  Detachment should occur within 3-10 minutes.  Do not agitate cells during this process as agitation encourages clustering of cells.  If cells are not detaching, place in incubator for 5 minutes to facilitate dispersal.
  3. Neutralize the trypsin-EDTA by adding complete media at a volume of 4x the volume of trypsin-EDTA added; centrifuge the cells and remove the supernatant
  4. Add 6-8 mL of growth medium to pellet and aspirate cells with gentle pipetting
  5. Add aliquots of the cell suspension to culture vessels at appropriate split ratio
  6. Incubate at 37°C in 5% CO2. Maintain culture at 2 x 103 and 1 x 104 cells/cm2. Count cells to ensure concentration does not exceed 7 x 104 cells/cm2.

A549 cells doubling time is approximately 22 hours.  Split confluent A549 cell culture 1:4 to 1:9 every 4-7 days with trypsin/EDTA and renew complete medium every 2-3 days.  For long term storage, freeze cells in a modified DMEM medium containing 20% FBS and 10% DMSO (as opposed to culturing medium using 10% FBS) with a cell concentration of approximately 2 x 106 cells/vial.

A549 protocol (UCSC) for cell propagation and handling – A549 PDF

Troubleshooting Subculture Procedures

Cell viability is low after passaging cells:

  • Cells were exposed to dissociation agent too long; only leave dissociation agent on cells long enough for cell detachment
  • Reduce harsh pipetting procedures during cell collection

If cells are difficult to detach from flask:

  • The cells were too confluent and cell-to-cell junctions are extremely tight preventing dissociation agents from reaching the cell interface; resolve this problem by subculturing the cells before they become confluent
  • Dissociation agent is too weak and a higher concentration must be used; incubating the flask containing the dissociation agent while waiting for detachment will increase enzymatic activity.
  • Ensure the flask was washed completely with sterile 1x PBS prior to addition of the dissociation agent; if necessary, two 1x PBS washes can be used.

If cells form clumps after detachment from flask:

  • Cells were centrifuged too hard; spin cells at 100 x g for 5 minutes to pellet
  • If cells are not immediately added to a new flask after dissociation, place the flask or vial on ice; this will decrease cell aggregation


  • Confluency: Much like Goldilocks, cells in flasks must be grown in an environment that falls within certain ranges. If there are too few cells in the flask, the cells will stop growing due to lack of cell contact.  However, too many cells in the flask will also cause the cells to stop proliferating due to lack of available room to grow.  In order to maintain cells in exponential growth, flask confluency should be maintained between 30-90%.
  • Thawed Aliquot: Freshly thawed cells are extremely fragile and should be thawed in less than 1 minute. Also, do not pellet the cells by centrifugation to remove freezing media.  Instead, the DMSO will be diluted by adding the thawed aliquot directly to a flask containing warm media.
  • Passage Number: Passage numbers should be recorded on the flask and not allowed to reach high numbers. Cells undergo genetic drift and other genotypic variations with high passage number.  The end result will be slow growing cells, hard to transfect cells and data sets that cannot be repeated due to the changing genetics of the cell.

Cell Sources

A549 cells are available from a number of sources, including ATCC and DSMZ.


  • The effect of green tea extract on lung cancer cells: A549 cells were exposed to green tea extract, and 2D gel electrophoresis LC tandem mass spectrometry was performed on the cells before and after exposure. This study identified 14 protein areas that changed in expression after treatment with green tea extract. These proteins were involved in growth, motility and apoptosis, indicating that green tea extract may have some anti-tumor properties. LINK: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775082/
  • RSV effects on chemokine secretion in A549 cells: This study exposed A549 cells (and other lung tissue-derived cells) to respiratory syncytial virus (RSV) and measured the activation of a selected chemokine. By measuring the activation, researchers were able to determine how RSV induced chemokine secretion, which may allow development of novel RSV therapies. LINK: http://www.jimmunol.org/content/161/2/1007.long