Hi
I have recently thawed out a new vial from atcc of phoenix ampho cells and put them into a T75 flask but 24 hours later the majority of cells have not adhered and are in suspension. I was wondering from people who have worked with phoenix ampho cells have your experienced this before? do you need to coat your flask/plate with some sort of extracelular matrix prior to seed to get the cells to adhere? or do the cells just take more than 24hrs to adhere?
I had issues with frozen vials of floating cells and then reattaching them. Thaw them in very low amount of media after taking out DMSO in high serum.
The Phoenix cell line is based on the 293T cell line - which is not particularly adherent - i.e. no need to use trypsin to detach that cells for passaging.If you are using tissue culture treated plates/flasks - this should be sufficient for the cells to adhere.
Have you checked whether the cells in suspension are viable? (i.e. collect the super natant with cells and check via trypan blue exclusion?). If they are not viable - then you have one possible explanation.
the freeze/thaw process can be hard on the cells and can lead to high levels of cell death. If the person before you freezing down the vial was not careful - then you will have low viability upon thawing. I freeze down the cells using the following recipe:
Freezing mix: 5 parts 20% FCS/DMEM (or RPMI) media, 2 parts 100% FCS, 2 parts DMSO (make in that order).
Resuspend cells in 20% FCS/media and then mix 1:1 with the above. i.e. 500 uL of cell suspension with 500 uL of the above freezing mix.
Other considerations might include the cells taking some more time to adapt to your particular media preparation.
If after 24 hours there are some cells adhering to the plate, I would pipette off the supernatant and carefully add fresh media to expand those which are left stuck on the plate.
All the best.
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