I am doing SeaHorse experiments (mito stress test) for NK cells.
I use cell tak to immobilize the cells to the cell plate. I follow all the instructions as provided in Agilent protocol for immobilization. But the cells detach upon addition of media after initial seeding and during the injections.
The pH of the NaHCO3 used for cell tak dilution is 8.0 as recommended.
I have also tried adding 1N NaOH half the volume of cell tak used to make sure that the pH is in the right range. The problem still persists.
Can anyone tell why this might be happening.
There can be many reasons for this, including but not limited to:
Temperature of media (needs to be 37*C - or as close to physiological relevance in your cell type).
As you mentioned, pH of media can affect this - most cell types require a pH of 7.4 (+/- 0.1), but this may vary in your cell type - follow you cell-type protocols).
I think the main reason could be the strength of suction and addition of media to the plate. I had very finicky cells that did not like to stick to the plate - we used several different plate coatings, but what helped at the end of the day was using proper temped and pH media, and very very slowly and gently removing some media, but not all media (leaving some in the well as to not let the cells dry out), and very slowly and carefully adding new media in. Also, although very tedious process, doing one well at a time can help so the cells do not dry out. If you wish to remove all media, this may take several times of diluting old media with new (rinsing - similar protocol as when you are "washing" the cells in preparation for running the assay).
Another reason cells tend to come off the plate is their level of stress - stressed cells tend to lift off even with the gentlest hand.
I'm sure there may be other reasons, these are just what I could think of off the top of my head and from my own personal experience.
Good Luck!
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