I fixed the adherent cells with 4% paraformaldehyde for immunostaining after culturing cells on gelatin coated well of 48 well plate. After fixation I covered cells with enough amount of PBS and put the plates in the refrigerator. How long can I keep the cells in these circumstances?
Dear Maryam,
I would recommend no longer than 3 days, otherwise the cells will detach. However, the best way would be to proceed as soon as possible. If you have to store your fixed cells, it might be worse to try methanol fixation. After methanol fixation, cells should be dried completely and can be stored @4°C up to one week.
Good luck!
Hi Maryam,
As Anne-Sophie indicates above, staining shortly after fixation is optimal, However, if the fixation of the cells is good, they can be stored for several days or even weeks at 4 degrees in PBS before staining as long as they are not allowed to dry out. Over a long time the quality of the cells will tend to decline, but we have been able to immunolabel cells successfully after storage for even a couple of months. The length of time you can store your cells before immunolabeling and still be good labeling, will also be affected by the stability and abundance of the antigens of interest and the quality of the primary antibodies. For example, stable abundant proteins (for example, tubulin in microtubules) would be more likely to withstand long storage and still yield good labeling, than proteins like plasma membrane receptor proteins, which are usually less stable and present only in low abundance.
Good luck.
Hi Maryam, I agree with the above comments in that you should process the samples as soon as possible. Please be aware that PFA will still be active at 4oC and continue to cross-link proteins. PBS will not inhibit PFA activity.
Dear
Fixation of cultured cells is usually achieved by simply discarding the culture medium and adding the fixative solution.
So is it important to note that fixation can be stored at room temperature for 24 hours or 5 to 7days at freeze temperature. It is very important to optimize fixation conditions since underfixation or overfixation may reduce or abolish tissue immunoreactivity.
Thank
Marius
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