I am new to CRISPR/Cas9 genome editing. Currently, I am carrying out CRISPR/Cas9 knockout of a gene and as part of the process, I carried out electroporation of RBL-2H3 cells with PX458 + gRNA recombinant DNA. My enrichment approach is to use fluorescent activated cell sorting (FACS) to obtain the GFP+ single cells and screen them for knockouts. However, anytime I carry out the cell sorting, all the cells die. Can anyone help me address this challenge and improve their survival? I used BDFACS Melody Cell sorter.
Again, your assistance would be appreciated if you can help me answer these questions;
1. Is FACS buffer recommended compared to 1x PBS for resuspending the cells prior to sorting?
2. Do I have to add extra serum to my collection medium?
3. Does staining the cells with APC live/dead stain prior to sorting affect the viability of the cells?
Cell survival is largely cell-type dependent. Easy for some cells (Hela) extremely difficult for others. Obviously, for single clones, the cells have to be able to be able to grow from single cells...but that may not be sufficient. Sorting is also very stressful, and you may consider on-plate selection using cloning rings (old fashionned) or automated systems, if you have access to them. Using conditionned media or extra serum may help cells recover post seeding. Look for methods using RBL-2H3 cells specifically. My experience with live stains has been positive, but they probably won't make a huge difference if your cells die post seeding. Good luck!
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