Flow cytometry is a pervasive tool to characterize just about anything in cell biology. From quantifying the expression of surface antigens, to determining the physiological changes in cells and everything in between, flow cytometry is as indispensable to a cell biologist as a knife is to a surgeon. Cell sorting is pivotal in enabling researchers to sort and characterize very small populations or even single cells, that otherwise are difficult to isolate by conventional methods.

Cell sorters are handled almost exclusively by facility managers or trained operators and rarely by end users themselves. In this two-part series (which aims to be more practical than technical), I will attempt to describe ways in which you, as a researcher, can increase your efficiency of sorting. The first part gives pointers on how to prepare cells for sorting and how to get along with the sorting process. The second part guides you on how to evaluate your sorted cells and the importance of having a healthy interaction with core facility personnel.

Preparing Yourself for Cell Sorting

Handling Cells and Reagents

Cells are babies and reagents are baby food. Handle them with extreme care to ensure that you don’t lose out on the initial cell count. Remember that starting with a good cell number is paramount in ensuring that you or the operator acquires enough events to set the gates and decide on the sorting strategy. If you know you have a low cell count, inform your operator beforehand. While you’re at it, coat your catch tubes with a paint of protein such as BSA or just culture media prior to the sort to help neutralize some of the electrostatic charges in the tubes, thereby preventing abrupt breakage of the cell droplet as it hits the catch tube.

Get to Know Your Lab’s, ‘My Precious’

Before even getting started, acquaint yourself with the instrument you’ll be sorting on. Talk to your facility manager to get a basic idea about the instrument configuration, the fluorochromes it detects, compensation settings required if any, the different nozzles and pressures it uses for sorting, the types of catch tubes it sorts into. Don’t forget any other special features you can take advantage of and plan your experiment around. Document this information for later reference. Be prepared for repentance if you make a great 15-color immunophenotyping panel for sorting that 0.01% cancer stem cell population, and start acquiring data, only to find out that the instrument cannot detect some obscure Alexa Fluor XXX.

Be a Mockingbird

As the adage goes, better be safe than sorry.  Perform a mock sort with control or stained samples for the cell type at least a day in advance so that both you and the facility manager/operator can discuss potential pitfalls and limitations of the strategy. It can also serve as a document template, so you can directly run actual samples without setting up everything from scratch. You also get to decide on the type of controls best suited for the experiment (negative controls, FMOs, isotypes etc.). Doing a mock experiment also allows the operator to correctly align the side streams for your type of catch tubes or multi-well plates so that the sorted cells don’t fall outside.

Two’s a Crowd

Flow cytometry is all about characterizing one cell at a time during laser interrogation. Having many doublets or clumps can seriously compromise your data and also can be detrimental to the sorter. Clumps clog the highly precise micro-nozzles through which the sample passes and the sorter might abort the process if it senses a clog. The stream can get disturbed and in worse cases, hit the high voltage deflection plates, leading to electric sparks. The sorter is usually the most prized possession in a research lab and facility managers are very protective about it. Proper dislodging and pipetting along with a touch of DNAase can help prevent clumps to a large extent. Unless you’re facing a cell drought, always filter your cells before loading in the instrument.

Reserve the Front Row in Time

Sorters are mostly housed in central core facilities to cater to as many scientists as possible and chances are that, you might need to liaise with your facility manager to ensure that you get to reserve slots for your experiment well in advance. Avoid last minute rushes and booking your slots very close to other slots. Always present yourself in time for your slot. This ensures that the operator gets enough time to start-up and QC the instrument before going ahead. You will also avoid a bad rep with the core facility guys, the benefits of which will become evident with time.

Keep Calm and Let the Sorter Do Its Thing

Now that you have (hopefully!) ensured the best sample preparation possible, relax and get ready to roll. Sit with your operator and have a quick overview before you actually insert your sample in the loading port.

Thou Shall Not Ask

Sorting parameters such as Efficiency, Conflict Rate, Conflict Counts, etc. and some of the stream related parameters are usually for the operators and the instrument itself to decide on the best course for sorting. Most modern sorters will pause sorting automatically until these values return to normal. Your operator will be constantly monitoring these values and can interpret them better than you can. Unless the operator pushes the red panic button, fret not.

Lay Yer Hands off Me

The sorter is a very sensitive instrument. For the love of Herzenberg, never, ever hold the bonnet of the sorter or accidentally hit the sheath fluid lines during the sort. Even slight vibrations cause the stream to go astray and hamper the sort process. Avoid blocking air/HEPA filters near the sort chamber. And, don’t talk directly in front of the instrument or touch the catch tubes/plates without first sterilizing your hands. Use 70% ethanol or IPA before touching anything.

This Is the Mask!

More often than not, end users simply don’t understand the concept of using purity, yield, and face masks. For most experiments, it is better to leave the default sort options alone unless you know what you’re doing. If you have very specific criteria, let the operator know in advance. Don’t even bother changing your mind midway during the sort.

Monitoring the Sort

If the sorting goes for extended hours, people generally overlook the volume remaining in the sample tube. Often, during extended sorting sessions and especially at high flow rates, the sample runs out and the sorter starts to aspirate air which is when hell breaks loose. During extended hours, always ensure that at least one person is monitoring the level of the sample in the tube. The same goes for intermittently checking the levels in the catch tubes.

In the next part of this series, I will discuss what you can do to check whether your sorted cells are fine and provide some tips to take care of them.

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