I thawed a tube of Calu-3 cells from ATCC, following product instructions. The flask I used was not coated with any substrate. I used EMEM with 10% FBS and placed it in the usual 37degC incubator with 5% CO2 and water pan. The next day I discarded floating cells. Since then, these cells have always appeared round and bright (see attached photomicrograph). They appear to be able to change positions, moving from middle of vessel to the sides. At first the numbers grew slowly but later it started dying. I have changed the media to DMEM with sodium pyruvate and 10% FBS. I had transferred floating cells to another vessel coated with collagen IV. I had also trypsinized one flask and transferred all cells to one collagen IV-coated well of a 12-well plate, and refreshed media every 3 days. Each day I waited for it to adhere firmly and resemble the clumps of cells I saw in the ATCC photomicrographs. But after a month, they are still round and bright. Now, they are starting to die off. Can anyone advice or suggest what I can do to rescue these cells?
Have you checked the viability of the cells after thawing? When kind of culture vessel did you use when you first thawed the cells? You may have a poor recovery after thawing.
We used a T25 flask to thaw the tube of cells from ATCC. Subsequently when we saw the cells were not adhering properly, we started using a 12-well plate. The photomicrograph is of one well in the 12-well plate. We have not done a cell viability test but at one stage the number of cells was observed to be increasing. Hence we thought it was a matter of time before they would adhere properly and develop the expected morphology. But the morphology remained the same until they started to die in most wells.
We coated our flask/plate with 10ug/mL collagen IV. Is this concentration too low compared to your protocol for substrate coating to support growth/adherence?
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