How to Separate Dead Cells from Live Cells in Suspension
It can be tricky to figure out how to separate dead cells from live cells in suspension culture. It first takes understanding all the elements at play. Then, you must decide which approach to take based on the specific needs of your lab.
Adherent Cell Culture vs Suspension Cell Culture
To get started, we want to distinguish between adherent cell culture and suspension cell culture so we can be clear on what exactly we are working with in terms of our media.
Adherent cell culture has long been the standard for most cell observation, monitoring, and experimentation. It calls for cells to be placed and grown on an unmoving surface to which they can affix and grow. Think of the classic petri dish with agar.
The upside of using adherent cell culture, and the reason it has been in place as the standard for so long, is that it makes for spectacularly easy observation.
Once in a petri dish or flask, both clear media, the cells can be easy placed under a microscope and observed at close range. They can be monitored over time, the dish or flask can be marked for positioning and growth. And so much more.
The downside of using adherent cell culture is that, as the cells reproduce and expand, they will eventually outgrow the substance on which they are growing. Upscaling this culture is quite tricky and requires a process of passaging the cells onto a larger space and closely monitoring them.
Enter suspension culture.
While originally suspension culture was used only for mammalian cells, known to grow quite rapidly and require room to expand, more and more microbiologists are finding suspension culture easier to work with and suitable for their purposes.
Virtually all cells can be monitored and grown in suspension culture.
Advantages of Cell Suspension Culture
The upside of cell suspension culture is that, as the cells reproduce and outgrow the space in which they are contained, upscaling is a simple matter of finding a larger vessel and more growth medium.
Of course, there is one major downside to using suspension culture, and that lies in observation. Often, you’ll need to either fix the cells in suspension through centrifuge and paraformaldehyde or you need an inverted phase contrast microscope.
However, once you have either of these, or both, in your bag of tricks, you can effectively monitor your cells in suspension culture just fine.
How to Differentiate Dead Cells from Live Cells
The next task at hand will be how to differentiate between dead cells and live cells in suspension culture. After all, you want to know how your cells are performing, which ones are viable, what percentage is viable, and which cells are dying.
Ideally, from there, you will want to figure out why those cells are dying, which is obviously a topic for another day.
To identify dead cells, you can use the most common method, which is to add a cell-impermeant DNA binding dye to your suspension solution. Classic examples of this solution are propidium iodide and dye from the STYOX series.
Once you add the dye, the dead cells, which no longer have the ability to fend off invaders, like dyes, will change color. The living cells, with intact nuclei, will not allow the dye to permeate the membrane and will not change color.
How to Separate Dead Cells from Live Cells in Suspension
Once you have identified the dead cells, you will then likely want to separate them so as to maintain a healthy, living sample that continues to grow.
You can make this separation in a few ways.
Centrifuge
The first and most obvious is to centrifuge your suspension culture. The key is to keep it low and slow so as not to kill any live cells in the process.
The standard is 400x g for 15 minutes.
Once the centrifuge is done, the living cells will still be floating in the liquid, and the dead cells will have flocculated to the bottom, where you can remove them.
Dead Cell Clean Up Kit
Another approach is to invest in a cell clean up kit which will use microbeads and binding buffers that will magnetically attach to cell debris and dead cells, which will separate them from the live cells and allow you to collect them.
Shake It Up
Finally, in the most rudimentary process, you can manually, gently, agitate your suspension culture for several minutes and then set it back down, waiting until the dead cells collect to the bottom of the vessel. This approach of course has its drawbacks as you cannot know for certain how fast or slow you are agitating. But it is a step in the right direction.
Oculyze Image Recognition Software Can Help
When you get to the point of monitoring your cells in suspension culture, rather than use the naked eye, even with a microscope, you can speed things up and eliminate human error by having an image recognition software do the analysis for you.
Our software has been successfully used by our partners for counting cells in suspension. Find out more about the Oculyze technology on our IAP page and if you also need a solution to save work hours and improve the accuracy of your results, contact us and we’ll be happy to find out the details of your specific needs and find a solution to attend to them.
Summing It All Up
In the end, suspension cell culture is breakthrough technology that allows cells to continue to grow and us to continue to observe them as they grow. This advancement in science means we can keep up with changing cells for much longer and with much more ease than we could do with adherent culture.
Separate the live cells from the dead ones in suspension culture is a matter of choosing between magnetic attraction and agitation. Either works perfectly well, so it is up to the scientist to choose a preferred method.
Oculyze is here to help.
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