So I have been trying to standardise a zebrafish single cell suspension for downstream sequencing. I am consistently getting decent viability if I measure it via syto 9 and propidium iodide staining using a commercial single use hemocytometer.
But whenever I try to use the logos Luna cell counter or any other automated cell counter which uses 1:1 trypan blue (0.4%) staining, I am getting really poor results for the same exact samples. Any insight would be much appreciated.
The difference in cell counts obtained from different methods could be due to several factors, such as differences in staining methods, equipment used, and cell size distribution.
Trypan blue staining is commonly used for live/dead cell discrimination and is based on the principle that live cells exclude the dye while dead cells take up the dye and become visible under a microscope. Syto 9 and propidium iodide staining can also be used for live/dead cell discrimination, but with different mechanisms. Syto 9 is a membrane-permeant dye that stains all cells, while propidium iodide is a membrane-impermeant dye that stains only dead cells with compromised membranes. Therefore, the staining mechanisms of these dyes differ from that of trypan blue, which could explain the differences in cell counts obtained from different methods.
Additionally, automated cell counters use different algorithms and image analysis techniques to identify and count cells. It is possible that the Luna cell counter or other automated cell counters are not optimized for zebrafish cells or for the specific suspension conditions used in your experiment, leading to inaccurate counts.
Finally, it is important to note that the size distribution of cells in the sample can also affect the accuracy of cell counts. Automated cell counters may not be as accurate for cells that are very large or very small, as they may not be properly detected or counted.
To troubleshoot this issue, you could try comparing the cell counts obtained from different methods for different dilutions of the same cell suspension to see if the discrepancies are more or less pronounced at different cell densities. Additionally, you could try optimizing the staining and counting conditions for the specific cell type and suspension conditions you are working with. If you are still experiencing discrepancies, you may want to consult with technical support from the manufacturer of the automated cell counter or seek advice from other researchers who have experience with zebrafish single cell sequencing.
- Badges
- Science method