Flow Cytometry Staining Considerations When Combining Intracellular and Extracellular Readouts

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This webinar recording features a presentation and discussion on flow cytometry experimental design. In particular, the focus is on staining considerations for multiplexing intracellular and extracellular readouts, including fluorophore choice, antibody titration, use of appropriate controls, and optimization of the fixation and permeabilization steps.

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Partial transcript:
Today, I'll be presenting the flow cytometry-focused panel of Learning Labs Live. Today, we'll be talking about some staining considerations when multiplexing with intracellular and extracellular readouts. So, historically, flow cytometry has been known to be very amenable to multiplexing, but largely, this has been limited to extracellular readouts.

It's really common to use panels of CD markers on mixed-cell populations and use that CD marker information to identify the different types of cells that may be present in your sample. You can also break it down by proportion of cells that fall into each subtype. Intracellular targets, on the other hand, have been largely studied by other techniques like Westerns or by microscopy-based techniques.

So why might one want to consider bringing in extracellular targets to a flow cytometry experiment? Intracellular targets regular lots of major cell processes like apoptosis, cell cycle progression, and others, By including both intracellular and extracellular readouts in a flow experiment, it will allow you to get information on not only what types of cells are present in your sample, but also, what each subtype is doing and how each individual subtype of cell might be responding to various stimuli. So performing this type of experiment, your same general multiplexing considerations apply, just the same types of things you'd want to take into account when working with a panel of only extracellular antibodies, namely, those things that will include prioritizing bright fluorophores.

So you want to make sure to, whenever possible, use fluorophores that are going to give you the best separation between positive and negative populations. And to be able to get a sense of how bright different fluorophores are, you might want to consider searching for staining index values.

And just to give an example of what it might look like to use a weaker versus a stronger fluorophore, here I'm showing some of our FLT3 products. A couple of them have been conjugated to weaker fluors, in this case, Pacific Blue and Alexa Fluor 700; whereas, one of them has been conjugated to a stronger fluorophore, Alexa Fluor 700, that's going to give you a better separation. So in blue is a negative cell line, and in green here is a positive cell line. The solid lines indicate cells that have been stained with the FLT3 clone, whereas, the dashed lines indicate concentration-matched isotype controls. So you can see that, between the blue and the green lines for the Pacific Blue and the Alexa Fluor 700, we're getting maybe a little bit over a log of separation between the negative and positive populations. But with the Alexa 647 conjugate, we're getting a little over two logs. And it's important to note that these samples were all run on the same instrument and the same concentration of that FLT3 antibody was used to stain our cells.

The same types of cells were also used in this experiment. So it's going to be especially beneficial when you're dealing with cells that express targets more on a gradient. So you've got low, medium, and high expressers as opposed to just a binary on/off. You're also going to want to try to minimize spectral overlap, because this makes compensation easier. This becomes more important as you've got more fluorophores in your panel. That'll happen as your number of readouts will increase. If you're only looking at, maybe, somewhere in the area of four to six targets at once, this is less of a concern. But if you're getting into the eight to 10-plus range, this is something you'll want to pay more attention to.

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