Tools | Cell sorting guidelines

• No training is needed for full-service sorting: our staff run the sorter for you.
• Reserve sorters in iLab, in 30-minute increments, and choose the type of service under “Pricing details”.
• Work out how long to reserve with the Sort time estimator. Debris and dead cells take sorter time too.
• Filter your cells through a 35 µm strainer right before the sort, and bring labeled collection tubes with medium in them.
• For a new experiment, run a pilot sort first.
• We sort samples up to BSL-2 only. Answer the biosafety questions in iLab accurately.

The core runs seven cell sorters. Optical configurations are on the Instrument configurations page. To check which sorter can detect your fluorochromes, use the Instrument Selector.

Cell sorting separates cells or other particles by their light-scatter and fluorescence properties. All our sorters are electrostatic droplet sorters, used for particles up to about 30 µm in diameter. Each one puts every particle of interest into a droplet and directs that droplet into a collection device: a tube, or a well of a plate.

A thin stream of sheath fluid (PBS) is pushed through a small orifice, the nozzle. The sample is injected into the center of this stream, which lines the cells up in single file (hydrodynamic focusing). The nozzle vibrates at 10 to 100 kHz, which breaks the stream into droplets. Droplets that contain a wanted cell are electrically charged and deflected left or right in an electric field, so that they fall into the collection device. All other droplets fall straight down into the waste.

Sort modes

Some droplets may contain two or more cells, sometimes a wanted cell together with an unwanted one. The sort mode decides what happens to those droplets.

Sort efficiency

Sort efficiency is the fraction of the wanted cells detected by the sorter that are actually deflected into the collection tube. In enrich mode it is practically 100%. In purity mode we aim to keep it above 75%. It improves when the cells are well dissociated and do not clump (see Sample preparation), when the sample contains little debris, and when the sample is diluted and run at a lower event rate.

For an in-depth introduction, watch The Art of Sorting, a webinar by Beckman Coulter.

A sort succeeds only if the target populations can be identified reliably. If your cells come from solid tissue, practice and fine-tune the dissociation first, and use a microscope to confirm that the cells are well dissociated and that there is little debris.

Then schedule a pilot experiment, to:

• confirm that the cell preparation and the staining are reliable;
• confirm that the populations can be identified and sorted with acceptable purity;
• estimate how much time is needed to sort enough cells for the real experiment.

Choosing the nozzle: high speed or regular speed

Aurora CS and FACSAria sorters can run in high-speed mode (70 µm nozzle, about 60 psi sheath pressure) or in regular-speed mode (100 µm nozzle, 18 to 27 psi). FACSJazz and FACSMelody (backup sorter, cannot be reserved in iLab) run in regular-speed mode only, with the 100 µm nozzle. FACSAria also has 85 µm and 130 µm nozzles, and Aurora CS a 130 µm nozzle.

As a rule, the cells should be at least five times smaller than the nozzle. Cells up to 14 µm across (lymphocytes, for example) can be sorted at high speed; cells up to 20 µm with the 100 µm nozzle. Larger cells deflect inconsistently, and some sorted cells never reach the collection tube.

Most sorts use the 100 µm nozzle. Any other nozzle needs extra setup, which can take up to 30 minutes. The figures for each mode are in Sorter specifications.

Sample preparation

• Check your cells under a microscope before you bring them: put a small drop on a slide. They should be round and abundant, with very few aggregates and little debris.
• Adjust the concentration to 10–15 million cells/ml for regular-speed sorting (100 µm) or 20–30 million cells/ml for high-speed sorting (70 µm). We can dilute cells that are too concentrated, but for practical reasons we will not concentrate them. If you have few cells, bring them in at least 0.5 ml.
• Keep the cells well dissociated. Any physiological buffer can be used, but cells that clump lower the recovery and the sort efficiency, and clog the nozzle.
• For cells that tend to clump, use a Ca²⁺/Mg²⁺-free buffer such as PBS or HBSS with low protein content (≤ 2% BSA). EDTA (0.5–5 mM) and/or DNase (20–100 µg/ml) help further; for pH-sensitive cells, add HEPES (25 mM). Standard culture media contain Ca²⁺, Mg²⁺ and serum proteins, all of which favor clumping.
• Filter the cells through a 35 µm cell strainer (Falcon cat. 352235) immediately before the sort, not earlier: some cells re-aggregate after filtering.

Collection tubes

How many populations each sorter can collect at the same time, by collection device:

Bring labeled collection tubes with enough cell culture medium or buffer to cover the bottom of the tube. Sorted droplets should land in liquid, not on dry plastic, and in medium that contains some nutrients. 100% FBS can also be used. All our sorters can also sort into multi-well (96-well) plates.

Sorting time

The time a sort takes depends mainly on how many particles have to pass through the sorter. The Sort time estimator works it out from your numbers and recommends how long to reserve your selected sorter in iLab.

• High speed, 70 µm nozzle: up to 20,000–25,000 events per second, or 72–90 million events per hour.
• Regular speed, 100 µm nozzle: we run the Aurora CS at 8,000 events per second (about 29 million per hour), and FACSAria and FACSJazz at up to 10,000 events per second (36 million per hour).
• Sample volume is a limit too: at most about 5 ml of sample can be run in an hour.
• Debris and dead cells count. The sorter has to analyze every particle, and debris takes as much time as cells. Samples with 25–50% debris or non-viable cells are common.

Part of the reservation goes to setting up the sorter for your project. For a one-color experiment, 15 minutes or less is usually enough. Add at least 4 minutes for each additional color: a five-color sort needs about 35 minutes for setup, single-color controls and compensation or unmixing. On the Aurora CS spectral sorters, large panels that were thoroughly tested beforehand can go faster than this rule suggests: recording the reference controls and unmixing a well-designed 20-color panel takes about an hour.

Purity check

To check purity, an aliquot of the sorted cells is run again on the sorter or on another cytometer. About 5,000 sorted cells are needed. We recommend asking the operator for a purity check. It is not done by default, because sometimes too few cells are sorted to spare an aliquot, and because it takes extra time. The purity of sorted cells is typically above 95%.

Giving your sort the best chance

• We cannot guarantee that every sort will succeed. Sorters are complex instruments that can fail unexpectedly and cannot always be repaired right away, and a sort gets only one chance: if something goes wrong, the sample is lost.
• If there is a problem with a sort, tell us right away. It is much easier to work out what went wrong at the time than days later.
• Whenever possible, bring more cells than can be sorted in the reserved time, and we will focus on sorting the number you need for the experiment. If there is no minimum, we run the samples in the order you give them and stop when the time runs out: tell the operator how much time each sample should get, or which samples come first. Samples prepared the same way can still yield different numbers of sorted cells, through small differences in preparation or in biology.
• Cells can be sorted in culture medium. But when the sort efficiency is low or the nozzle keeps clogging, resuspending the cells in a cation-free, low-protein buffer can improve the result dramatically.
• Watch your sort, at least while the gates and the sort logic are set, and then for several minutes to see the sort rate and the sort efficiency and decide whether anything needs adjusting.
• Keep records of how you prepared the cells and how each sort went. Many of our users have improved their results considerably by using those records to refine their cell preparation.

We offer three types of cell sorting service. All are reserved in iLab; you choose the type under “Pricing details” when you make the reservation (see Reserving a sorter in iLab).

Trained users who are not yet confident running the sorter on their own, or who expect to need a lot of help, should reserve full-service.

If you reserve full-service

• On some sorters, reservations cannot be made over lunch (12 to 1 PM).
• Bring the cells to the lab yourself and stay for the setup, to confirm the gating strategy and the sort logic. You are welcome to watch the sort, at least for the first 5 to 10 minutes after sorting starts, to check the sort efficiency and the sort rate for each target population.
• Pick up the sorted cells when the sort is finished.

• No training is required for full-service sorting on any of our sorters: a member of our staff runs the sorter for you.
• To learn to sort in semi-assisted or unassisted mode, start with The Art of Sorting webinar by Beckman Coulter. It explains concepts you need to know before hands-on training.
• Training sessions are reserved in iLab like any other reservation, with the use type “Training”, and are charged at the full-service rate.
• Basic sorting skills on Aurora CS, FACSAria and FACSJazz take two training sessions of about 2 hours each (4 hours in total). On Aurora CS and FACSAria, one 2-hour session may be enough if you already know the software: SpectroFlo for Aurora, FACSDiva for FACSAria.
• FACSAria training takes place on FACSAria 2 (FACSAria II) in ARC, or on FACSAria 3 in MCRI for researchers who work in the Morgan building. Training on either qualifies you to sort on all three FACSAria sorters.
• Advanced features and troubleshooting common problems need additional training. We suggest reserving your first sort after training as full-service, so that a member of our lab can guide you through it.
• FACSMelody (backup sorter, cannot be reserved in iLab) is very user-friendly. When it is in service, it can be reserved in semi-assisted or full-service mode, and users with flow cytometry experience need only a 30-minute training session at the start of their first sort.
• Written training guides for each sorter are on our home page, under Instrument Training Guides.

1. Get an iLab account Register in iLab if you do not have an account. Questions about registering go to coresadmin@chop.edu, or ask any member of our lab.

   Not at CHOP? To find our core, log in to iLab, click the menu icon 1 at the top left, choose “Core Facilities” 2, select “Cores at partner institutions” or “Cores at other institutions” in the “View” list, and look for the Flow Cytometry Core at CHOP.

   Show screenshot

   
2. Set up a payment method A form of payment must be active in iLab before you can make a reservation.
   • CHOP researchers: ask your PI or business manager to give you access to an active payment account in iLab.
   • Penn researchers: set up a PO. Your business manager may need our BenBuys details: Phila 200, Site 49513, Children’s Hospital of Philadelphia.
   • All other researchers: set up a PO from your own institution, or pay by credit card.
   Questions about payment: coresadmin@chop.edu.
3. Choose the sorter See Our sorters, the optics on the Instrument configurations page, and the Instrument Selector for which sorter can detect your fluorochromes. If you need help choosing, email flowcytometry@chop.edu.
4. Work out how long to reserve Use the Sort time estimator. Sorters are reserved in 30-minute increments.
5. Open the sorter’s calendar Log in to iLab, open the “Reserve instruments” tab, scroll to the bottom of the page and click “Sorters”. Click “View schedule” for the sorter you want, then click and drag over a free slot in the calendar.
   Show screenshot

   
6. Fill in the reservation The numbers match the screenshots.
   • 1 Answer “Is this reservation for training?” and fill in the questionnaire, including the biosafety questions.
   • 2 To change the time, click the pencil to the right of the end time, then the green check mark. This does not save the reservation.
   • 3 Click “Pricing details” and choose the type of service: Full-service (untrained users, business hours), Semi-assisted (trained users working on their own, business hours), Unassisted (trained users, after hours) or Training.
   • 4 Choose the fund, account or PO number for this reservation.
   • 5 Click “Save Reservation” at the bottom left.
   Show screenshots

   
7. Wait for approval The reservation shows as “Needs approval”. This is normal; we will contact you if we have questions. Our staff approve reservations just before the start time. Once a reservation is approved, it can no longer be changed in iLab, though it can still be cancelled.

Come to the room of the sorter you reserved (see Our sorters).

Getting into our labs

What to bring

• Filtered cell suspensions in 5 ml FACS tubes, including single-color compensation controls, unstained cells and FMO controls if you need them.
• Labeled collection tubes containing cell culture medium or FBS.
• About 10 ml of sample buffer, for diluting the cells if needed.
• Extra collection medium and tubes, in case new collection tubes have to be prepared.

Before sorting starts, check with the operator that all gates are set correctly and that the populations to be sorted are clearly identified.

The Flow Cytometry Core is a BSL-2 facility. We cannot sort any agent that requires containment above BSL-2.

• All our cell sorters are enclosed in class II biosafety cabinets and can be used for samples that require containment up to BSL-2.
• Under current CHOP policy, all cells of human origin, including established cell lines, must be handled at BSL-2 or higher.
• The iLab questionnaire for every reservation includes biosafety questions. Answer them accurately: we will cancel a reservation if there is any doubt that the samples can be handled safely.

The main characteristics of our sorters in regular-speed and high-speed mode.

Email flowcytometry@chop.edu. Messages reach every member of the Flow Core, so you will usually get an answer sooner than by writing to one person.

For anything urgent that needs immediate action, call 215-590-3402 or talk to a member of our lab.