Sorter FAQs

1. What is flow sorting?

Sorting is defined as the separation of particles or cells of interest from other particles or cells in the sample. The FCCF in Bonn has four different cell sorters that work by electrostatically deflecting charged droplets that ideally contain the cells of interest. Each sorter is capable of sorting four or even six populations simultaneously.

2. Which sorters do we have?

A basic overview of each sorter is given below. The setup of each sorter can be altered to a degree depending on the cell type and fluorochromes used therefore it is essential we know the details of these in advance.

BD FACSDiscover™

5 laser (349nm, 405nm, 488nm, 561nm, 637nm)

78 fluorescence APD detectors

>40 colors simultainiously

6-way sorting

bulk and single-cell sorting

index sort

nozzle size: 85µm, 100µm, 130µm

BD CellView™ Image Technology

BD SpectralFX™ Technology

biosafety cabinet

BD FACSChorus Software

BD FACSAria™ Fusion:

5 laser (355nm, 405nm, 488nm, 561nm, 640nm)

18 parameter

4-way sorting

bulk and single-cell sorting

index sort

nozzle size: 70µm, 85µm, 100µm

BD FACSDiva Software (v 9.01)

biosafety cabinet (S2 approved)

BD FACSAria™ III:

5 laser (375nm*, 405nm*, 488nm, 561nm, 640nm) *alternatively

20 parameter

4-way sorting

bulk and single-cell sorting

index sort

nozzle size: 70µm, 85µm, 100µm

FACSDiva Software (v 9.01)

Sony MA900™:

4 laser (405nm, 488nm, 561nm, 638nm)

detection parameters: 12 fluorescence + 2 scatter

sort devices: 2-way tube, 4-way tube, multiwell plates, PCR plate

bulk and single cell sorting

sorting chip size: 70 µm, 100 µm, 130 µm

easy-to-use easy-to-learn software for operator-free sorting 24/7

3. What do I need to know when booking a sort?

Although you will be able to view the Scheduler to allow you to plan your experiments, sorters cannot be booked by anyone other than employees of the FCCF. (except MA900™) There are two reasons for this. Firstly we need to ensure that someone is available to perform your sort and secondly we need the details of your sort so that we can set up the sorters accordingly. So in consultation with a member of the CF, you will let us know the following information:

- Which fluorochromes you are using

We need to optimize the optical filters used and the choice of fluorochrome will also allow us to set up the correct lasers. We will also be able to judge if there are likely to be problems and it may allow us to suggest alternatives.

- The cell type to be sorted and the size of your cells

This influences the size of the nozzle we use in sorting. As a rule of thumb, the nozzle should be at least 4 times the diameter of the cells to be sorted. Therefore in practice, this means that small round cells (lymphocytes, splenocytes, thymocytes) use a 70 μm nozzle and epithelial cells or fibroblasts will require a 100 μm nozzle. Occasionally this will be determined empirically with your cell type. A practical consequence of having to use a 100 μm nozzle is that sorting will not be as quick (see section 4). Having the right size nozzle with respect to your cells helps give better side streams, prevents clogging of the nozzle, and increases purity and yield. The size of your cells can be estimated using a viability analyzer such as the Countess or a "Neubauer" counting chamber.

- Will the sort be aseptic?

We will routinely clean all fluid lines and the sort chamber with alcohol, bleach and detergent to sterilize the cytometer. This adds to the setup time needed.

- How many cells can I bring?

This is VERY important! An appreciation of what the sorters are capable of is essential for setting the right level of expectation about what can be achieved in the time available. The following table shows how many events are possible under different setup conditions on each sorter.

All sorters: (if nozzle/chip size is available, see above)

70µm

20.000cells/sec = 72 million/hr

85μm

12,000cells/sec = 44 million/hr

100µm

6,000cells/sec = 22 million/hr

130µm

3.000cells/sec = 11 million/hr

These numbers assume the standard sheath pressure used for each nozzle, if this is changed, the rate will also change. Each situation may be different so these numbers should only be used as a guide to help you calculate the number of cells to bring for the amount of time you have booked. The sort operator will give you a rough estimate of how many cells you can expect once the sorting has started. The numbers quoted in the section above are events processed above any threshold. In your sample, this will include: whole live cells, dead cells and debris. If you know the relative contributions of each, you can work out pro rata approximately how many cells you will get back. An appreciation of this can help avoid potential sources of argument. Although increasing the threshold will make small particles/debris invisible to the machine, they are still in your sample and will be sorted with your cells. This can cause major problems if you are sorting for future PCR or sequencing so in general, we keep the threshold as low as we can while maintaining as high a throughput as possible. Although each sort is potentially different with its own specific challenges, the FCCF staff can adjust running conditions appropriately to optimize efficiency, purity and speed under concideration of sorter induced cell stress (See point 5 for further details.)

4. What do I need to bring on the day of my sort?

- How should I bring my samples to the Lab?

In either 5 ml or 15 ml Falcons. Cells should be in an appropriate buffer that preserves viability and controls pH (HEPES buffer will help reduce pH changes that are induced by the high pressures used in the cell sorter). They can be in medium or FACS buffer (PBS with 1% BSA or up to 10% sterile filtered FCS) Please make sure that your sample tubes are labeled. We will also need collection tubes (generally 5 ml or 15 Falcons but we can use Eppendorfs and culture plates as well) which should contain medium. The amount of medium will depend on how many cells you will collect. For small numbers, it is useful to have a full tube to avoid cell loss. Collection tubes can have 100% sterile filtered serum or be coated with serum to help prevent cell loss. Also bear in mind that the cells will come back in sheath fluid which is FACSFlow or PBS.

Each drop using a 70 μm nozzle has a volume of approximately 2nl so a 5 ml Falcon tube containing 500 μl medium will be full when approx 3 million cells have been collected. With a 100 μm nozzle we get around 1 million cells per 5 ml tube.

- How many cells will I get back?

There is no firm answer to this as it will be influenced by the initial percentage of the target population, how many populations you want to sort, the sort mode used and the sample preparation itself. You can help with the last one – make sure you have a good single-cell suspension, no clumps, little debris and few dead cells. Clumps may be removed prior to sorting by filtering through a 100µm mesh. Cell re-clumping can be minimized by sorting at 4°C or by adding EDTA and/or DNAse to the medium. Dead cells can often be excluded by their light scatter characteristics. However, this isn’t always the case so we advise adding a DNA-binding dye that can be used as a dead cell exclusion marker. There are several dyes that can be used: DAPI, Hoechst33258, Propidium iodide, 7AAD and TO-PRO-3 are the most common. It is also a good idea to have an idea of the number of cells you will require for any subsequent assays.

- Any other practical tips?

The use of polypropylene rather than polystyrene tubes for collection particularly if you anticipate low cell numbers in your population of interest may be beneficial. Polypropylene tubes build up less static charge and so fewer cells are repelled.

- Cell concentration?

5 x 10^6 /ml for large cells, 2 x 10^7/ml for small cells.

- What are ‘sort modes’?

These are how we tell the sorter what stringencies we want to apply to a sort and they can be altered depending on whether we want to optimize purity, count accuracy, or yield. All flow cytometers interrogate cells by passing them through one or more lasers and collecting the emitted fluorescence. Sorters such as the Discover, the Aria and AriaFusion and the MA900 work by ejecting the cells in a fluid stream through the orifice of the nozzle. By applying high-frequency vibration to the nozzle, drops are formed which we can then deflect by applying a charge to them. To sort the cells of interest we need to know the time it takes for a cell to pass through the laser (at which point we know what its fluorescence characteristics are) to the point at which individual drops start to form. This is known as the drop delay and is set by the operator prior to the sort. By knowing this and by correlating it with the appearance of cells through the laser we know where each cell is situated in the ejected column of fluid and hence in which drop it will be. In an ideal world only one cell will be in a particular drop. However, in reality the arrival of cells is a stochastic process and there can be more than one cell per drop. If a wanted cell is accompanied by an unwanted cell, we can’t sort the drop as it would reduce purity. Sometimes a cell is situated near the edge of a drop and could be either in the expected drop or the neighboring drop. In these cases, we would sort both drops (assuming a contaminating cell isn’t in either of them). Cells can be lost in two ways. Firstly, if they pass through the laser too close to be separately analyzed (we call these hardware aborts). This happens if cells are clumpy, big in relation to the nozzle size or too concentrated. Cells will also be lost if they can’t successfully be sorted - for example where a drop contains a cell of interest and also an unwanted cell, to sort this drop would compromise purity so it is normally not sorted – we call these co-incidence or software aborts. These practical considerations will affect your yield. The Aria uses a complex series of sort masks and which aim to sort as many cells as possible with as few aborts as possible and does so by sorting one or more drops if it can. We can also perform an enrich sort. This would be applicable if you have a precious or small population. In this situation, all cells that qualify will be sorted even if there are unwanted cells nearby in the stream. So, purity is reduced but yield is maximized. Enrich sorting can only be performed for one population. The final mode is called Single-cell mode. This is used when count accuracy is paramount, for example when cloning, it uses a very stringent mode to ensure that only one cell is sorted. Again, this can only be used for a single population at a time. The sort mode used will be discussed with you.

- Gating strategies/sorting gates

It’s your sort, we will do it as you want. However… the FCCF staff has a lot of experience in identifying and excluding debris and dead cells, we are experienced in machine set up including filter selection and fluorescence compensation. All gates and regions will be discussed but are ultimately your responsibility. The sorter will sort what you tell it to sort, it’s up to you to give it the right instructions and you should be satisfied about gating before the sort begins.

- What about my booking time?

There is some degree of flexibility. We realize that sometimes preparation takes longer than expected or there is a problem of some type. However, it may not always be possible to extend your booking time. Another sort may be booked – and we need to allow at least 1/2 hour between sorts to clean and prepare the sorter – or the sort operator may have another commitment. If you are going to be late, please call, it’s a simple courtesy that will save heartache. Likewise, if we experience a problem that would delay your start, we will call you and let you know.

We do not cross-charge for time so we can afford to sort slowly to get better yield but remember it’s a balance between that and making sure all users get a fair chance. We cannot guarantee extended hours without advance booking! Sorts are scheduled on a first-booked-first-served basis. However, we also reserve the right to move sorts between machines in the event of breakdown. If your sort has to be moved or cancelled, we will liaise with you as soon as we know and endeavor to re-schedule as soon as possible. We will attempt to give priority to sorts that are time dependent e.g. on animal availability, but we expect a reciprocal degree of flexibility from our users. If you book to finish at a specific time and you are not done, we reserve the right to terminate the sort in order to minimize consequent effects on the running schedule.

- How do you assess the success of a sort?

Good question! There are three metrics that are important: Purity, Recovery and Yield. Purity is probably the one that most users are concerned with. It should be possible to get 95%+ purity. Purity is defined as the number of cells in your sorted tube that fulfil your sort criteria. A small aliquot can be re-run on either the sorter or an analyzer to assess this. Note: adding new viability dye might be necessary to assess the number of dead cells after sorting. Recovery is the ratio of the number of cells in your sorted tube relative to the number that the cytometers says it has sorted. This should be around 80%. There will be some cell loss due to cell death and not all sorted droplets going into the collection tube (another reason to avoid polystyrene collection tubes). It must be stressed that samples should NOT be spun or manipulated before counting Yield is the percentage of cells you get back relative to the amount in your sample. Yield will be 70%+ in most cases but will be influenced by the sort modes used. If there are problems with any of these, please let us know. Unfortunately stream in air sorting isn’t perfect and neither are we (almost though) so please let us know if there is a problem subsequent to your sort. This could be low viability, poor cell number, contamination or some other problem. If we don’t know, we can’t do anything about it and we could potentially affect other people’s sorts. Reduced purity and recovery are almost certainly due to factors other than the sorter itself. We can help troubleshoot these problems but only if we know about them. Please take the time to provide feedback on your sorts.

5. Sorter induced cell stress (SICS)

All the available cell sorters in our facility are pressurized, dropletbased systems, which apply significant environmental pressure and shear stress to cells during sorting. Recently, the flow cytometry community has become increasingly aware about the potential negative effects this could have on sorted cells and the term “sorter induced cell stress” (SICS) has been proposed. Following publication investigates this phenomenom in more detail: An evaluation of sorter induced cell stress (SICS) on peripheral blood mononuclear cells (PBMCs) after different sort conditions - Are your sorted cells getting SICS?

6. Other information

To comply with German Health and Safety policies, you need to be aware that all the sorters in the FCCF work by electrostatic drop deflection and are therefore always producing aerosols. This is particularly important if there is a clog in the nozzle as drops of even smaller size are produced. For this reason, all human samples and some retrovirally infected cells MUST be sorted at S2 level. This means that you will be limited to the Aria Fusion™ which is housed in a biosafety cabinet and located in an S2 laboratory. All S2 related work must be announced beforhand to the operators. If in any doubt, speak to a member of the FCCF staff. All sorters except the Aria III are housed in a biosafety carbinet, ensuring a sterile and contained environment suitable for work with primary human samples, including peripheral blood or other potentially biohazardous materials. Please read our safety guidelines before starting your work at the core facility.

7. Lab Contacts

All the members of the FCCF are proficient in the alignment and set-up of the cell sorters. There is a range of experience in terms of multicolor experiments and cell types used. Experience is gained in the real world by sorting your samples and by attending conferences and vendor trainings. In some cases, for example, where there is a scientific collaboration, one operator may perform the bulk of a user’s sorts. However, in most cases, you are likely to deal with any member of staff. If you have any concerns, please bring them to the FCCF managements attention.

Core Facility Manager: Andreas Dolf

email: andreas.dolf@uni-bonn.de

office: 0228/287-11028

mobile: 0170-3750882

Flow Cytometry Scientist: Dr. Andreas Pannek

email: apannek@uni-bonn.de

office: 0228/287- 11023

lab: 0228/287-51349

Flow Cytometry Technician: Peter Wurst

email: pwurst@uni-bonn.de

sorter: 0228/287- 11024

lab: 0228/287-51349