I have a cell line that is stably expressing eGFP and mRFP1. I want to introduce another color that will be easily distinguishable by flow cytometry. I was suggested to use AmCyan which has an excitation peak of 457 nm and emission peak of 491 nm, and is usually detected by the violet laser. So, I used the lenti viral vector expressing AmCyan1 from Addgene (Plasmid# 48974) and stably transduced my cells. The 293T cells that I used for making the virus and the cells of interest both emitted green fluorescence under the microscope (GFP filter), which I thought could be due to spillover into the GFP channel. So, it suggested that the transfection and virus production were fine. However, when I tested them by flow cytometry, it is very strange that I could not detect any AmCyan positive signal in both of these cell lines, but I do see GFP+ expression in the cell line of interest. Has anyone used this plasmid before and encountered this problem? I even got my plasmid sequenced and it completely gets aligned to the AmCyan sequence. My sole purpose with this experiment is to use a third marker that can be easily distinguished from GFP and RFP by flow. Does anyone have any suggestions with the AmCyan problem or has anyone used any other fluorescent protein in flow in conjunction with GFP and RFP?
Hello, here the detail using tandem tagged mCherry-EGFP-LC3 (C-G-LC3) to quantify autophagic flux in single cells by ratiometric flow cytometry and to isolate subpopulations of cells based on their relative levels of autophagic flux. This method measures autophagic flux rather than autophagosome number and is an important addition to the autophagy researcher’s array of tools for measuring autophagy. Two crucial steps in this method are i) generate cells constitutively expressing C-G-LC3 with low to medium fluorescence and low fluorescence variability, and ii) correctly set up gates and voltage/gain on a properly equipped flow cytometer. We have used this method to measure autophagic flux in a variety of cell types and experimental systems using many different autophagy stimuli. On a sorting flow cytometer, this technique can be used to isolate cells with different levels of basal autophagic flux, or cells with variable induction of flux in response to a given stimulus for further analysis or experimentation. We have also combined quantification of autophagic flux with methods to measure apoptosis and cell surface proteins, demonstrating the usefulness of this protocol in combination with other flow cytometry labels and markers.
See also below link.
https://www.researchgate.net/topic/FACS-Staining
Good luck!
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