MDCK is good morphology condtion in DMEM when passaged in our lab shows slightly different and bigger in DMEM prepared by us. Its difficult to find out why. We don't use CO2 during incubation and is it making this difference?
Thank you Barbara Ridolfi. But I have a doubt about it. Is it must to provide CO2 for MDCK growth?
You can add HEPES buffer to your medium to control the pH and will not require CO2. Make sure you keep the caps tight.
Please let me know your culture medium components are. Also, could you provide a picture of your cells?
WE add DMEM one packet, 10ml vitamin soln, 10ml antibiotic- antimycotic soln, 2g sodium bicarbonate into 1 lit of HEPES solution and after filtration (0.2micro) we add FBS 10%.
WE add DMEM one packet, 10ml vitamin soln, 10ml antibiotic- antimycotic soln, 2g sodium bicarbonate into 1 lit of HEPES solution and after filtration (0.2micro) we add FBS 10%.
Yes this is been a problem for quite some time. Even the image that ATCC shows in their website are not a complete monolayer and looks like a population of more than one cell type. You can start finding gaps in the monolayer after few passages, which sometimes makes difficult for determination of virus titration. You can try with cells from old stock.
ATCC MDCK cells may also contain a mixed population. Are you trying to isolate or propagate influenza virus in your cells?
Thank you Mr. Teluguakula Narasaraju, I will try reviving from Old stock.
Mr. Thomas Rowe : I am trying to isolate and cultivate influenza virus in MDCk
Are you having difficulties isolating and cultivating influenza viruses in your cells?
Please contact me directly if you are having difficulties with your cultures
ok sir. Please let me know if any commercially available kit for checking mycoplasma contamination.
dmem low glucose, 10% donor bovine serum, 1% antibiotic 1% insulin, 5% co2, and we use until 100-120 passaged, the co2 its used for the pH control.
Thank you Oliver Schildgen, Orlando vargas-Sierra for the suggestions.
Since you do not use CO2/ bicarbonate buffered media, I assume you use Hepes. I have found from experience some cells do not grow well with this buffer. Also the comment on mycoplasma is pertinent and you should test for possible contamination.
Good luck with your research
Thank you @Jerome Tanner. My cells are growing well now with 10% FBS and DMEM without Co2 incubation. Thank you for your suggestions.
MDCK cell line can go further into several passages and they are very strong cells compared with other types of cell culture like PHTBE cells .... If you do not use 5 % co2 you must add HEPES to the media (1/100 dilution) ..... also try to use DMEM gibco 500 ml bottle with addition of 5 ml pen/strept and 5 ml HEPES and 0.5 ml Amphotrocin B and 10% FBS ...... that is it ..... I did notice bigger size before when I used antibiotics several times to cells to retrieve them from bacterial contamination ... the cells were so weared take long time to grow and show bigger shape ... so either you do not have the correct media or have bacterial contamination or used too much antibiotics that is toxic to cells ....
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