3 Considerations To Ensure Your Cell Sorting Flow Cytometry Experiments Run Smoothly

Written By: Tim Bushnell, PhD

Cell sorting is such an important technique because it’s a gateway tool for many other downstream applications.

Assays such as culturing cells, genomic analysis, proteomics, injections into mice and the like are enabled by cell sorting, allowing researchers to use a homogeneously defined population of cells in their experiments.

With single-cell genomics we can readily characterize a population in great detail, but we have to have a single purified population for it to work.

That’s where the cell sorter comes in.

There are all sorts of applications we can do with a purified population and with the advances in sorting and fluorescent technologies, it is possible to isolate complex phenotypes from rare populations.

Fluorescent proteins are a great example of being able to look at the expression of the inter se or the marker coupled to a fluorescent protein so we can sort.

We can do two or three or four of these GFP, YFP and some of the fruit fluorochrome so you can look at the expression patterns of two or three different proteins at the same time using expression cassettes.

Then you will be able to isolate cells and have one of the express proteins two or all three.

Great technology.

But in order to reap the benefits of this technology, you need to consider a few things about cell sorting first…

1. Size dictates almost everything you are going to do.

The first important thing about cell sorting to remember is that the cell size dictates almost everything you’re gonna do.

And when I talk about cell size, I’m talking about the cell volume, not the flattened out measurement you can make on a microscope, but the volume of the cell.

Now, this is a cell, this is a nozzle for a cell sorter here. And these come in different sizes ranging from 70 micron to over 130 microns.

Figure 1: Picture of a nozzle from a cell sorter.

So what does that mean?

Once I know the volume of my cell and if you assume a cell spherical 4/3πr³, it’ll give us the rough volume that also gives us the radius.

We want to use a nozzle that is four to five times larger than our cells.

So if I have a lymphocyte at 10 Micron diameter, a 70 Micron nozzle will work. If I’m looking at an astrocyte that’s 30 Microns in diameter then I’m gonna really have to be looking at something at 130 Micron range and there are several tables out there that show this.

Figure 2: Relationship between nozzle size and cell type.

Now, once you determine the nozzle size, the sort operators are gonna then set up the instrument and that nozzle size is gonna dictate what the sheet pressure is gonna be.

The larger the nozzle the lower the sheet pressure.

Figure 3: Relationship between nozzle size, sheath pressure, and frequency.

That, in turn, dictates the frequency of droplet generation because there’s a balance between the frequency of droplet generation and the sheet pressure.

If you know the drop frequency of droplet generation you can determine how many events per second you want. So when we express the droplet generation, it’s talking about tens of thousands of droplets per second.

Based on Poisson statistics that we’ve talked about in several blogs here and on Facebook, we want to use an event rate of one event per four droplets. So, if the system is generating 90,000 droplets per second, the event rate needs to be about 22,500.

With a larger nozzle, this may mean sorting at only 4,000 or 5,000 events per second.

With everything set up on the instrument, it is possible to do the back of the envelope calculation to determine how long the sort should take.

Figure 4: Sorting calculations based on population frequency, and frequency of droplet generation.

Estimate how long it’s gonna take to run your sample based upon that speed, based upon how many events you need and based upon the frequency of the population.

There are tables that we’ve published that show that those types of calculations.

2. Sample preparation is key.

The second important concept is sample preparation.

A clog will ruin your day. When the nozzle clogs, it will force the system to shut down. If you’re fortunate, it is a minor delay, and only requires a minor delay in the sorting process. With very bad clogs, this may take hours to clean and get the system back up and running.

So filtering the sample, just before you put the cells on the sorter is a critical final step in sample preparation.

Second, think about what the preparation of your cells is going through.

If you’ve got adherent cells and you’re going to be using trypsin to pull them off the plate, you don’t want to just put serum back to neutralize the trypsin because you’re gonna add back all the compounds Calcium, Magnesium that the cells need to start to adhere again.

Also add a little bit of DNAase, about 10 U/ml because that will help minimize the amount of free DNA causing cells to stick together. In using DNAse for years to help reduce clogging, I have never seen any impact on genomic analysis

So remember these three steps for sample preparation: Filter the cells, use soybean trypsin inhibitor and add DNAse.

3. What type of tube are you collecting your cells in?

The third issue to review is the catch tube. What are your sorted cells going to rest in until the end of the sort? The catch tube is very important.

With electrostatic sorters, the droplet containing the cell is charged, and if the angles are off, it is possible for the droplet to be attracted to the side of the tube, and therefore the cell would die as the droplet evaporated.

To prevent this, coating the tube’s negative charge is one way to minimize this effect. To do this, add your staining buffer to the tube and let it sit for half an hour, 40 minutes or longer, just so that any protein in the staining buffer can coat the plastic and reduce the charge.

The other thing to optimize in the tube is the catch buffer. Using 100% serum isn’t a great idea because those early cells are gonna be sitting in a 100% serum for a while.

Using your staining buffer with twice the amount of protein as you would usually use is a good place to start. However, test different conditions to ensure the best for keeping the cells happy is critical.

With the myriad of downstream applications that are improved after isolation of cells. Taking the time to optimize the experiment to ensure that the resulting cells are of the highest quality for the downstream application. Knowing the cell size will determine the rate at which the cells could be sorted. Knowing the best tips in sample preparation including filtering the cells before sorting, adding DNAse to the sample will help reduce clumping caused by DNA, and using trypsin inhibitors for neutralization of trypsin used to remove adherent cells all help minimize clumping. Finally, making sure that the catch tubes are treated properly to reduce the chance of the droplets sticking to the side while ensuring that the catch solution preserves the cells.

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Tim Bushnell, PhD
Tim Bushnell, PhD

Tim Bushnell holds a PhD in Biology from the Rensselaer Polytechnic Institute. He is a co-founder of—and didactic mind behind—ExCyte, the world’s leading flow cytometry training company, which organization boasts a veritable library of in-the-lab resources on sequencing, microscopy, and related topics in the life sciences.

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