Dear all,
I have issue with cells detaching during PFA fixation and washes.
Following a paper's instruction we are differentiation neurons from iPSC and then plate them on PDL+Laminin coating, grow for 10-20 days (long term limit 1 months as per paper) but when we PFA fix and wash them cell detach all togheter like a sheet of cells. Collaborators use the same coating with primary mouse neurons without problems, I know iPSC-derived neurons would be more immature but still the paper do not report issuse about attachment.
Does anyone have any suggestion on why they detach and how to solve this probem?
PBS wash is with same PBS previously used with cells and PFA is 4% in PBS.
Thanks in advance.
How long are you fixing for (I do 10 mins without issues)? Also, make sure that the PBS/PFA solution you have is not cold but at RT. Alternatively, although not ideal, if you think that your cells are not fixed properly because they detach, you could fix with PFA (16% or 32%) and dilute it to obtain 4% in your cell medium by adding it drop by drop.. I have done this and they fix normally but prefer to use the 4% solution when I can.
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