Immunolabeling of intracellular proteins typically requires fixing/permeabilizing your cells they will not be viable afterwards. Additionally, typically FACS staining is done with primary antibodies(because you can use multiple from the same host) but it can be done with a secondary ones as well.

Regardless of the above to get the highest purity you would probably want to sort with a different channel for each target to get the best resolution between positive and negative peaks. If you use the same channel your peaks will naturally clump due to single positive and double positive cells with weaker intensity, never mind the possibility that you could have a nontarget cell population that stains very brightly(compared to your target cells) for one of your proteins in your triple positive population.

Okay great, thank you for your help!

From this I assume that you conjugate a different fluorescent marker onto each antibody. Can you explain what you mean by channels?

Sorry I have no experience of using FACS myself and am trying to understand it better!

Alice Thomson FACS is hard to master, but quite easy to understand. If you can, just take a couple of hours one day and go to a FACS machine, ask the technician to explain to you the basics and, trust me, you'll understand way better than I or other people can explain over the internet.

Until then...as William Suslovic said above, intracellular (and also surface) markers in FACS usually need just one antibody (improperly called "primary" since there is no secondary to follow). You may be more familiar with ELISA techniques where many protocols use a secondary antibody on top of the primary.

https://www.thermofisher.com/ro/en/home/life-science/cell-analysis/fluorophores/alexa-fluor-700.html

You can see here the characteristics of a fluorochrome.

The AlexaFluor700 fluorochrome is usually excited using the 633 (red) laser. But there can be also other fluorochromes excited by the red laser in the same experiment. Therefore, in order to separate the signals from these fluorochromes, there are multiple filters and detectors assigned to the red laser (see the attached photo). In the example, both AlexaFluor700 and APC are excited by the same red laser, but they are strategically chosen so their peaks of emission are distanced enough to allow you to have detectors on top of each. As you can see, the emission spectra of AF700 and APC overlap, but the filters for the detectors (660/20 and 730/45, respectively) are strategically chosen as to

1) capture as much of the emitted light as possible (filters are on top of the emission peaks)

2) minimize the overspill of light from one fluorochrome into the other's detector.

The selected filter+detector is what we call a channel. So, back to your question, if you had 3 distinct antibodies, but all 3 had the same fluorochrome, they would all emit light at the same wavelength and be captured by the same filter+detector...so they would all end up in the same channel.

CASE 1.

If the intracellular expression of those 3 proteins would be roughly equal, you would just get 3 overlapped signals because

1) they are all captured by the same channel

2) they all have the same intensity and therefore can't be differentiated based on luminosity.

And it would look like this: __/\__ (3 peaks on top of eachother)

CASE 2.

If the intracellular expression of those 3 proteins is different, but not so much (say for example: protein 1; protein 2 = 1.2 x protein 1; protein 3 = 1.5 x protein 1), then you would have 3 distinct peaks, but very close to eachother, like

__/\'\'\__ (3 distinct peaks, but partially overlapped)

CASE 3

Best case scenario: the intracellular expression of those 3 proteins is very different (say for example: protein 1; protein 2 = 4 x protein 1; protein 3 = 8 x protein 1). In such a case, you would have 3 distinct signal peaks in your channel such as

__/\_/\_/\__ (3 distinct peaks in the same channel)

However, even then, you would never know which peak comes from which antibody. Maybe, you could find in the literature that Protein 3 > Protein 2 > Protein 1 in that type of neuron, but even so...It's just not scientifically correct to do this.

That is why in FACS we use antibodies conjugated with distinct fluorochromes and I recommend you use distinct fluorochromes for each of your target of interest.

Also, i would like to add that there are protocols where the same fluorochrome is used for multiple antibodies. I used an intracellular staining kit for cytokines: IL-2, TNF-a and IFN-y. We had antibodies against each of these 3 cytokines and they were all labeled with the fluorochrome PE.

What we did was to split our cells in 3 and then we single-stained each part with PE-anti-IL-2, PE-anti-TNF-a, and PE-anti-IFN-y.

It works as, in the end, you would be able to say that x% of your cells are IL-2+, y% are TNFa+, and z% are IFNy+....

BUT (big but)

You can never say how many cells are double positive (IL2+TNFa+ or IL2+IFNy+ or IFNy+TNFa+) or triple positive (IL2+TNFa+IFNy+)...which is a big downside for the experiment. Having distinct fluorochromes for each of your antibodies enables simultaneous detection of multiple markers which is the whole idea of FACS.

Ion Bogdan Mănescu

Thank you! I really appreciate the time you took to explain It for me!

It all makes so much more sense.