Hi, currently I am trying to isolate nuclei using the "10xGenomics Sample Preparation Demonstrated Protocol". The protocol suggested varying the lysis time to see which one is the most optimal and it suggested assessing the nuclear isolation using hemocytometer.
It is only possible to access dead vs living cells, and it is hard to specifically identify enucleated cells. To address this, I tried to use double staining using DAPI and WGA. This double staining method is not so successful as it is still fairly hard to distinguish between enucleated VS intact cells, even under confocal.
May I ask suggestion to troubleshoot this? The cell that I use was HEK293 cells.
A standard approach to verify nuclei isolation (or to check the integrity of cytosolic versus nuclear extracts) is to detect proteins that are restricted to the nucleus, such as histones, using antibodies.
You can double-label with Acridine Orange and Propidium Iodide. Both can permeate the nuclear membrane (PI is live cell membrane-impermeant, but does penetrate nuclear membranes). If you only see the AO, it indicates an intact cell membrane, but PI quenches AO when when both are bound, so PI labeling can indicate either dead but unlysed cells, OR intact isolated nuclei.
You can use trypan blue staining as shown in data of following kits:
https://inventbiotech.com/search?type=product&q=nuclei+isolation
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