I have a problem - A collaborative lab sent me Calu-3 cells by mail but they appear to have slow growth. The flask is in the incubator for more then 2 weeks and there is less then 50% confluence. The cells divide but most of them remain detached in the medium and are removed during the medium replacement.
The medium used is: DMEM:F12 with 10% FBS, 1%L-glutamin, 1% NEAA and 100 mg:ml penicillin/streptomycin. How long normally do Calu-3 cell line take to get confluent?
Last time the cells have it... but this time no mycoplasma...
What more can it be?
Caryotype alteration
Did you verift Mycoplasma with PCR test?
You have to check Lot of your serum and DMEM
Find the new sample of cells or make the cloning of your cells by serial delution method in 96-well plate, take several single clones separately and observe the behavoir during cultiuvation.
Susana, when we got frozen cells , the correct is to put them in a medium with higher serum concentration. Also could you ask who gave you these cells to know the time of growth.
The cells was not frozen when I got it. The person that gave me the cells say that cells normally grow in 10 days... but this ones are in the flask for almost 3 weeks and are not confluent yet...
Are you growing them in the same flask that they sent? Few people seal the flasks that they wont leak during the transfer!
In the day that the cells arrive most of it was not attached so I let in the same flask for 2 days and then washed with PBS and added trypsin and transfer it to another flask.
You may collect cells after trypsinisation and put them in "a smaler surface" well (ex. the well of 6-well plate) with 20% of serum to be sure to have a good number of cells to start cell proliferation
Be sure that there are no mycoplasma, you have really exclude this option. Mycoplasma-contaminated cells have proliferation problem-they grow well, afterwords they almost stop to grow, then they may start to grow again. But you may have alterated response to the stimulation in mycoplasma-contaminated cells
You might want to check the media. This could happen if your media is expired or if the CO2 percentage is not proper in the incubator.
Yes I will see with other media from other lot that a colleague that will borrow me some tomorow. I think can be it. The 4 bottles that I have are from the same lot and the first 2 didn't work. Lets hope that is it...
It was from the medium... I changed the medium to DMEM and borrow some of DMEM/F-12 from a colleague (both from sigma) and the cells in that medium grow.
My Gibco medium Ref: 31330 and Lot:879664 probably was out of order and I was wasting my time with it... Does anyone had problems with Gibco mediums?
Thanks everybody for the help
Now the cells seem to be growing :) lets see...
I am culturing Calu-3 and I face the same problem, they are not attaching to the culture plates. My question is what did you do when you change the media? what about the cells which are on the media?
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