In my previous article I discussed steps you can implement to ensure that a sample is ready for cell sorting. But now it’s time to make sure the sort worked. Here are a few sorting checks and measures to ensure that all’s well that ends well.
Post-sorting Checks and Measures
Re-evaluate Your Catch Tubes
Sorting is strenuous not just for you, but also for your cells. After the laser interrogation point, a lot of factors, such as nozzle size and pressure, temperature of the catch buffer or plain bad luck, can damage your cells before reaching the catch tubes. Therefore, it is important to re-run the catch tube as a sample to ensure that the cells fall in the corresponding sort gate. However, this might not always happen since you already excited the tagged fluorophore and it might not possess the same peak emission by the time you analyse your catch tube. So slightly relax your sort gates to help you heave a sigh of relief.
Fragile! Handle With Care
Cells that have just come out of the nozzle are as vulnerable as a dodo at the edge of a cliff. Don’t get all worked up and take them for a spin just yet. Allow them some time to settle down. Cell sorters are usually housed in air-conditioned rooms wherein the room temperature hovers between 19-22°C. Your cells are not used to wearing thermals so better allow them to come to a cosy 37°C before toying around with them.
Statistics to the Rescue
Strange as it may seem, the best yardstick for a sorter’s performance is its post-sort recovery than purity. Andrew Riddell and colleagues have come up with a unique parameter called Rmax that can help calculate the sort recovery based on several parameters. They have also designed calculators which can help you calculate the Rmax value for your particular sort. Facility managers might benefit more from this but it pays to be educated about it as an end user.
You Can Still Enrich
Sometimes, despite the best optimization, you may end with far less purity than desired. One way to get around this, is to put the cells back in culture and allow them to grow to a sizeable population before re-attempting the sort. If you already know that you have insufficient counts of cells, grow them in culture till they are in suitable numbers and try to enrich for the target population with a magnetic sorter. Then, continue with FACS to get better purity and/or yield.
Getting efficient sorts requires a lot of skill – both on part of the operator as well as the end user. What I have described are just some of my observations as a facility manager having had the experience of manning a great core facility.
Have you ever entertained the idea of learning to program? Have you tried but felt discouraged by the overwhelming amount of information out there? If you answered yes to both of those questions, I encourage you to try again with the following resources. Computer science is one of the best subjects to self learn. …
The flow cytometer that we have all grown to know and love may have only come into its own in the 1990’s, but who would have known that the first cell sorter was invented as early as the 1950’s? With the recent death of one of the key developers of fluorescence activated cell sorting (FACS),…
No matter how we make measurements, there will be variation (a spread of data). Take 100 people and ask them to guess your age and you will get a range of results: some will be too low (excellent!), some too high (not so good!). It is the same with any of our laboratory experiments –…
If you were to peek into a protein biochemist’s bag of tricks, what would you find? A mortar and pestle for collecting samples, some columns for isolating proteins and a mass spec instrument? Perhaps. But what about those little eppendorf tubes full of enzymes and helpful molecules? Certainly, each scientist has his/her own favorite. Here…
Every biochemist is familiar with proteases. More often than not, proteases cause a lot of anxiety. To this end, a lot of research has been done in developing techniques to prevent the activity of proteases. But some of these proteases can be the good guys too! For example, you can use them to separate your…
10 Things Every Molecular Biologist Should Know
The eBook with top tips from our Researcher community.
