If the 3 lasers can distinguish 3 kinds of samples/organisms I would say in theory yes, the issue you may have is that Chloroplast may auto-fluoresce with a red colour that may match the Nile Red stain and provoke some confounding effects. I recommend you give it a try by using the 3 samples separately, from which you can define the "gates" of the facs for each population. Then, prepare mixed solutions using various known concentrations of each samples, and see if you observe the same proportions (%) of separation at the output of Facs in each gate. Another issue is that you should do the experiment right after staining to avoid to loose cells/samples or to avoid the staining to degrade itself. is it clear?

Hi Samuel, unless your lab uses Nile red frequently to assess lipid accumulation in multi-stain assays, I suggest avoiding it. Nile red exhibits solvatochromism in a hydrophobic environment (such as neutral lipid), and this causes a 40nm emissions to lower (more blue/green) wavelengths. As a result, it overlaps heavily with green fluorophores. I've worked a lot with Nile red (but not with algae) and I know it will be detected in FL1 of your cytometer. Depending on how much lipid (hence staining) is present, you may or may not be able to compensate the signal from Nile red out of the SYTO signal. If you have the money for it, you can use other BODIPY or LipidTOX dyes that emit in the red and stain lipid.

In terms of actually doing the experiment, Arno has good suggestions. To check for spectral overlap of the fluors, you can use samples stained individually with each fluor to set the compensation parameters (your flow technician should know how to do this).

When you process your data, I suggest using FlowJo. You need to buy it , but its very good software. I havent used them, but free softwares are Flowing and CyFlogic.

Hope this helps!

Here's a paper for you on Nile red:

https://www.ncbi.nlm.nih.gov/pubmed/4031658

https://www.hindawi.com/journals/ijs/2016/5215086/cta/