I have isolated successfully protoplasts from Arabidopsis roots following the published article (Ryu et al., 2019). I am just wondering if there is a way to somehow check its efficiency because I needed these cells for scRNA-seq. Thank you a lot.
I usually count cell numbers and do Evans blue staining to check cell viability after protoplast isolation. I use a hemacytometer for counting cells because sometimes cell counter give wrong numbers of live cells and dead cells. So I check cells under the microscope and count live cells and dead cell after staining. I use cells their cell viability is over 75% for scRNA-seq and cell density is over 1,000 cells/ul.
Kook Hui Ryu hello, thank you for the information. But my question is about the efficiency not the viability. I wanted to know how would you know if you isolated protoplasts, like cells from outer to the innermost core of the root (e.g. if you get same cell proportion of each of the cell file)?
Unfortunately, I don't know whether I get whole cell population or not before getting the final result of scRNA-seq. I got most population of cells from Arabidopsis roots, but I couldn't get same proportion of each cell files from other plant species. To figure out this problem, I check remaining tissues after cell sorting. If many cells are not isolated from remaining tissues, it means that population of isolated protoplasts doesn't include all cell files. I'm still testing to figure it out.
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes