I am not familiar at all with FACS but I would like to use it to isolate cells from a tissue and then do RNAseq of the sorted cell populations. These cells are expressing either only RFP (single-positive), or RFP and GFP (double-positive), or RFP,GFP and CFP (triple-positive), or nothing (triple-negative). Is there a limitation in sorting them in one go?
The only limitation is the machine you are using. If the machine can sort 4 different tubes (sort 3 and 1 negative tube) and has the lasers/filters for these fluorochromes, you should be able to sort all at once.
Just adding some details to Steven Mathis's answer. For sorting all of them in the same time, besides you have to use FACS machine that can do 4-way sorting, your machine has to be equipped with violet laser (405nm or 440nm) and blue laser (488nm) and or green-yellow laser (561nm or 550nm), and also equipped with band pass filters according to their max emissions.
Max excitation/emission for CFP is 430nm/485nm, for (E)GFP is 488nm/509nm and for RFP is 555nm/584nm.
Hello Steven and Eko. Thank you both for your answers. I am glad it's feasible and I will make sure I find a machine with these specifications for my experiment..
Make sure that you have good compensation controls to detect specific signal for each fluorescent protein. You need strong single positive signal for each fluorescent protein and a negative signal with the same autofluorescence characteristics (usually parental cells, untransfected cells or negative population). Good luck!