I am doing calibration curves using CyQUANT Direct Assay to 10 different pancreatic cancer cell lines. I've noticed that the fluorescence of some cell lines double at the same cell number. Is this due to different amount of DNA or could it be due to an error in the procedure during cell count or seeding? I am not sure if CyQUANT can bind to RNA. If that is the case, I would expect RNA expression also affecting the fluorescence.
I am using the same settings and instrument for the reading.
Since the CyQUANT assay detects the DNA in live cells, differences between cell lines may reflect differences in the ability of the dye to accumulate in the cell lines, rather than (or in addition to) differences in the amount of DNA between them. A likely reason for differences in intracellular dye accumulation between cell lines is differences between them in the activity of multidrug efflux pumps such as MDR and MRP in the cell membrane.
I agree with both answers. I might also suggest that you adjust the gating for each cell line you are using. As well, consider the fact that lines that are more mitotically active may have more dna content as well. You can assess this later point by pulsing the lines with cfse or other marker and then looking for number of divisions after a period of time.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line