The golden rule of flow cytometry, especially cell sorting is: ‘Put good cells in and get good cells out’.
When you sort you might not get good cells out and you may not get the numbers you were expecting. In my previous article I touched on a few reasons why your cell numbers might be low after sorting, and in this current article I mention a couple more reasons why.
As discussed in the article on how a sorter works, sorting has little effect on your cells. However, if they are poorly to start with, then they are unlikely to survive sorting.
It’s important to use a viability dye such as propidium iodide or DAPI when sorting, so you can determine the viability of the cells as they go into the machine. These dyes only go into cells with a compromised membrane so your healthy, happy cells won’t take up the dye and be affected. Your dying or dead cells will take up the viability dye and will show positive staining. Using a viability dye might clue you in to why you don’t get as many cells back.
Yield and recovery
Your yield and recovery factor into the number of cells you get back after a sort. And your purity will determine how successful the sort was. But these terms can often be confused. Let’s get them straight and talk about how they affect your cell numbers.
The purity of your cells is the percentage of cells of interest in your collection tube after they have been sorted. Purity is ascertained by running an aliquot of sorted cells back through the machine after you have finished your sort.
It will show if the required populations to be sorted have been sorted. You can also add a little bit of viability dye and determine the post-sorting viability.
Note: When checking the purity, you should start with your original gating scheme but, you may need to alter the gates slightly due to quenching/photo-bleaching of dyes and/or some osmotic and other changes.
Cells can often be sorted to above 95% purity, although with a distinct population that is being sorted, this can be above 99%.
Purity = Proportion of cells of interest/total number of cells in the sort tube
The yield of cells is the number of cells of interest in your sample tube compared to the number of cells of interest in your sorted tube. This number will be much lower, as you will lose cells during the sort due to aborts.
Yield = Proportion of sorted cells of interest/ total number of cells of interest in initial tube
Recovery of cells is the number of cells that are in your collection tube compared to the number of cells that the sorter tells you are in the tube. It is usual to lose cells do to various reasons:
- Cells moving out of droplets and subsequent sorting of empty drops
- Cells sticking to the tubes
- Cells dying during sorting
- Sometime cells just leap out of the tubes!
After sorting you should expect around 70-80% recovery of your cells.
Recovery = Number of cells in sorting tube/number of cells that were sorted
Improve your yield and recovery
It is possible to improve the yield and recovery of your sorts.
- Start with a nice sample preparation. Healthy, clump-free cells will give the best results.
- Sort into collection media containing a protein source to help with recovery. A protein source such as fetal calf serum or bovine serum albumin dissolves the charge on the droplet and helps to stop cells sticking to the tube.
- Use polypropylene collection tubes to keep cells from sticking to the sides of the tubes. Polystryene tubes (often used for analyzers) will hold the charge and you will have lower cell numbers in your tube.
As a general rule, if your tube contains 1 million happy healthy cells, then expect to get around half of those into your sorting tube. You might get more, but it’s always better to start with more cells than you think you will need.