Hello everyone,
We're working on the cardiomyocyte nuclei isolation from the snap-frozen heart, and we are still facing some problems when it comes to determining if they're good/bad nuclei under brightfield microscope. Does any of you have pictures of how good nuclei should look like after isolation? And do nuclei always have to be in good round shape to be considered good? We take a look at 10x genomics pictures about good nuclei (https://kb.10xgenomics.com/hc/en-us/articles/360020348651-How-can-I-assess-the-quality-of-my-nuclei-for-Single-Cell-ATAC-or-Single-Cell-Multiome-ATAC-GEX-Sequencing-) but we see it differently every time we lyse the tissue, so we are not sure what are the real nuclei and how to evaluate whether they're good or not.
Attached is the picture we have the other day when we check the suspension under a 40x microscope, but we are not sure if the round white dot is the nuclei. Can someone please help me to verify this?
Thanks a lot!
Use a higher magnification, ideally.
Nuclei should be ~10um in diameter, so going by the TINY scalebar in the corner of your images, you're in the right ballpark.
You can also use a nuclear dye (hoechst/DAPI) and stick them on a fluorescence microscope: if they're nuclei, they'll be really obviously blue.
Isolation of nuclei from cardiovascular tissue is pretty tough. Usually, the yield is low but a good isolation method should give you good number and morphology as shown in following link:
https://inventbiotech.com/collections/single-nuclei-isolation
A recent paper shows excellent data on single nuclei RNA-seq using cardiovascular tissues:
https://www.nature.com/articles/s44161-022-00140-7
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