Has anybody else observed or figured out a way to address the washing away of pyroptotic/de-attached macs during all of the wash steps required for immunofluorescence? I still see cells that fit the needs of most of my experiments, but I know that I'm washing away a significant portion of cells that have come off the substrate during the process of pyroptosis.
depending on your readout you can use z-vad or caspase-1-/- cells to protect cells from pyroptosis. z-vad or caspase-1 deficiency do not inhibit asc oligomerization. however, if you are looking for caspase-1 downstream events you have to determine the latest timepoint where the cells are still not detaching.
reference: The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.
Thank you for the answer, Fabianno. I had considered using an inhibitor (YVAD-cho) to essentially synchronize cells at the point of inflammasome assembly prior to death and even did so for some TEM work, but I didn't continue with it for the thought that maybe it was considered messing with the system too much.
Is it common for inflammasome immunofluorescence to include an inhibitor? I hadn't thought so.
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