Research Flow Cytometry Core

Frequently Asked Questions

Can I use GFP and YFP simultaneously while sorting or analyzing my cells?
Yes, although GFP and YFP emission spectra greatly overlap, it is possible to detect both fluorescent proteins with a specific set of filters. Using 510/20BP - 495LP filters for GFP and 545/35BP -525LP filters for YFP minimize the spectral overlap between these two fluorescent proteins.
For sorting, can I bring my cells resuspended in medium containing FBS?
The best sorting media is 1x PBS with 0.5% of FBS and 1mM of EDTA. However, depending on the needs of your cells you may increase the concentration of FBS up to 2%.
I need to sort cells and isolate RNA, should I collect my cells in TriZol or another buffer to isolate RNA?
We do not recommend collecting cells in TriZol buffer, collecting cells in RLT buffer (Qiagen) or 1X PBS has given the best results so far.
How do I discriminate between live cells and dead cells when doing flow cytometry analysis?
Discrimination between viable and non-viable cells can be carried out with the use of the 7-AAD or PI (Propidium Iodide). Both dyes will label the non-viable cells by binding to the nuclei of those cells. The nucleic acid of viable cells will not be accessible to the dye and will not be stained. When analyzing the data collected, gate out cells stained with the viability dye.