Recently, I started to culture A549 cells. The cell looks very well at the other day after thawing, but it grows extremely slowly while culturing. They become large and flat and look like senescence. I thought it might be mycoplasma contamination, but the medium is clean and without any visible black dots. And also I added ciprofloxacin to the medium to inhibit mycoplasma, but it is not helpful. I tried both 1640 and DMEM medium supplemented with 10% FBS, 10mM Hepes, 1% PS (according to the experience of other labs, both medium is suitable for A549). There is no improvement. How can I change this situation? Does it need any additional supplement, cytokines or growth factors?
How confluent are your cells after thawing?
I made the experience that cells grow incredibly slow/stop growing when they are too "lonely".
Hi, Sophie Ziegler. The conflunce was about 30% after the cells attached the plate. It costs about 1 week to reach about 80%-90% conflunce. And I could split the cells just 1:1 to 1:2, then another 7-10 days is needed untile the next splitting. Thus I think it may not just because of "lonely".
Thanks Kar. Are the FBS, PS and HEPEs Supplements sufficient or are additional supplements needed?
Thanks for sharing your experience and your suggestion, Daniel. I will try horse serum!
Hey,
how is the growing of your cells now?? We use them a lot in our lab and we cultivate them in DMEM (4,5 g glucose) with 10% FBS and 1% Pen/Strep thats all and they grow very fast. We don´t add any additional supplements. If we are not careful we have to split 2 times a week 1:10
Thanks, Sabine Schröder. Currently my A549 cells grow fast. I thawed and dropped several times of A549 cells. Finally, I found that there were some cells which sticked to the plate after trypsinized, and I maintained these cells. This cell population grows better and better. During this period, I also changed 1640 and FBS from another company, without additional supplements. Thus, I have no ideal that which is the key factor that changed the situation of my cells. However, there is still one thing that these survived A549 cells morhpologically looks a little different from the original A549 cells.
Best regards!
Try using F-12K Medium, Catalog No. 30-2004+ fetal bovine serum to a final concentration of 10%.
Cell density should be between 2 x 10>3 and 1 x 10>4 viable cells/cm2. Do not exceed 7 x 10>4 cells/cm2.
https://www.atcc.org/products/all/CCL-185.aspx#culturemethod
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