Hi colleagues, I'm having problems sorting neural crest cells. I isolated NCC from the head of E13.5 embryos marked with tdTomato. The procedure for the isolation of the cells includes some steps using TrypLE for embryo digestion, followed by FBS supplementation, cell scraper to reduce clogs during sorting and I keep them on serum-free DMEM for sorting. Sorting takes ~20min and the cells are collected in DMEM supplemented with 10% FBS. I usually get 6 million td+ cells for 4 embryos.
I incubated them in 6-well plates coated with 2% gelatin and changed the media the next day, but almost 60% of them were dead. Does anyone have any tips on how to improve the cell's viability or if there is anything I should do to improve the sorting?
**This is the 3rd time I'm planning to do and previous lab members couldn't figure it out.
Dear Julia Raulino Lima,@
I understand that you are experiencing issues with neural crest cell (NCC) viability after cell sorting. Based on the procedure you described, I have a few suggestions that might help improve cell viability and the overall success of your experiment:
1. Reduce TrypLE exposure: Prolonged exposure to TrypLE can be harmful to cells. Try to minimize the exposure time by monitoring the digestion process and stopping it as soon as the cells are dissociated.
2. Gentle dissociation: During cell dissociation, apply gentle mechanical force using a pipette to break up cell clumps instead of the cell scraper. This may reduce cellular damage and improve cell viability.
3. Optimize sorting conditions: Ensure that the sheath fluid, nozzle size, and pressure settings on the cell sorter are appropriate for NCCs. This can minimize damage during sorting and enhance cell viability.
4. Add ROCK inhibitor: Supplement the DMEM media with a Rho-associated coiled-coil kinase (ROCK) inhibitor, such as Y-27632. This can improve cell survival and attachment, particularly for stem cells and primary cells.
5. Use a lower passage number: Cells at a high passage number may be more prone to stress, leading to reduced viability. Consider using NCCs with a lower passage number to increase their chances of survival.
6. Optimize gelatin concentration: The gelatin coating concentration may affect cell attachment and survival. You may want to test different concentrations of gelatin (e.g., 0.1%, 0.5%, 1%, and 2%) to find the optimal coating level for NCCs.
7. Monitor the incubation environment: Make sure the incubator is providing the appropriate temperature, humidity, and CO2 conditions for NCCs. Any deviations from optimal conditions may impact cell viability.
8. Assess cell health before sorting: Check the health and morphology of the NCCs under a microscope before sorting. Unhealthy cells may contribute to the high death rate after sorting.
I hope these suggestions help improve the viability of your neural crest cells after sorting.
Best regards,
Mahdi Hatami
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA