Is it true that cells tend to become 'stickier' and clump easier after fixation (e.g. with PFA)?
I also find that my PFA-fixed cells do not pellet down as well after centrifugation for the washing steps?
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#2
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Posted 28 November 2011 - 07:42 PM
Cells are not necessarily stickier upon fixation, but cells that are already clumping together will become permanently stuck together upon fixation. the PFA crosslinks proteins in close proximity. Whether these are proteins on a single cell's surface, or proteins in close proximity between two different cells is of no consequence.
Regarding the pelleting issues. It's likely you're fixing your cells in the presence of a buffer containing protein (such as BSA or FCS). Make sure to wash your cells with plain PBS before PFA fixation. Just like on the cells, the PFA will crosslink proteins in the buffer making the solution denser and therefore the cells don't pellet as well.
Regarding the pelleting issues. It's likely you're fixing your cells in the presence of a buffer containing protein (such as BSA or FCS). Make sure to wash your cells with plain PBS before PFA fixation. Just like on the cells, the PFA will crosslink proteins in the buffer making the solution denser and therefore the cells don't pellet as well.
#3
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Posted 29 November 2011 - 03:28 AM
RynDggn, on 28 November 2011 - 07:42 PM, said:
Cells are not necessarily stickier upon fixation, but cells that are already clumping together will become permanently stuck together upon fixation. the PFA crosslinks proteins in close proximity. Whether these are proteins on a single cell's surface, or proteins in close proximity between two different cells is of no consequence.
Regarding the pelleting issues. It's likely you're fixing your cells in the presence of a buffer containing protein (such as BSA or FCS). Make sure to wash your cells with plain PBS before PFA fixation. Just like on the cells, the PFA will crosslink proteins in the buffer making the solution denser and therefore the cells don't pellet as well.
Regarding the pelleting issues. It's likely you're fixing your cells in the presence of a buffer containing protein (such as BSA or FCS). Make sure to wash your cells with plain PBS before PFA fixation. Just like on the cells, the PFA will crosslink proteins in the buffer making the solution denser and therefore the cells don't pellet as well.
Thats makes perfect sense. I fix my cells in 4% PFA made up in PBS so definitely protein-free. It could be some carry over from the growth medium or washing medium (which has BSA). The pelleting issue only starts after the primary and worsens after secondary. Could it be some PFA were leftover in the buffer?
