University of Dundee

Cell Sorting

Cell sorting at SLS is provided as a service to all researchers, but requires the assistance of a trained operator. The cell sorting schedule can fill up quickly, so you are advised to make an appointment at least a week in advance. It may be possible to accommodate some emergencies at short notice, but this can in no way be guaranteed. If for any reason you need to cancel a booking, please inform us as soon as possible so that your time-slot can be made free for someone else. You should aim to meet with the operator several days prior to the sort to plan your experiment.

What type of cells do you want to sort?

Safety issues: both chemical and biological hazard information for your sample is very important before sorting can be performed. The process of cell sorting purposefully produces single cell aerosols in order for the process to work, and the lack of any form of containment means that both the operator and surrounding environment are exposed to these aerosols. We will need information such as; from which species do your cells originate? Will the cells be live or fixed? Into what biohazard category do they fall? Does the sample contain a chemical hazard?

Physical properties: in order to optimise the sort conditions, we need to know how big and how fragile the cells are. During sorting, cells are passed under high pressure through a narrow orifice. Larger cells will require a larger orifice, and particularly fragile cells may not survive higher pressures. Both orifice size and pressure dramatically affect the rate at which cells can be sorted, and hence the length of your sort. The general rule is: the larger and more fragile the cells the slower the sort speed.

Sterility: if sterility of the sample is necessary (i.e. If the sorted cells are going to be cultured on) it is possible to perform the sorting procedure under 'asceptic' conditions. We do not have the facilities to achieve a truly sterile environment, so it is always advisable to culture cells after sorting in the presence of antibiotics. It is also important that samples are sterile when they come for sorting. [note: antibodies and fluorescent dyes are a common source of contamination!!].

How will your sub-population of cells be identified?

Before cell sorting is undertaken it is necessary to perform preliminary flow analysis to establish the flow cytometric profiles of the cells. These investigations will help to establish what fluorophores/dyes are going to be used to identify the cells of interest.

It will also tell you the frequency at which the cells of interest occur in the total cell population (see the section on 'how many cells do i need?). If researchers are not familiar with flow cytometry as a technique, then we can perform these preliminary investigations for you.

How long will your sort take?

The speed at which cell sorting can take place depends on the type of cells to be sorted. As a general rule the larger and more fragile a cell the slower the sort rate. Also, cells that have a tendency to clump (i.e. Adherent cell lines and transiently transfected cells) will be more difficult to sort. Conversely, cells such as thymocytes that are small (<10 μm diameter) and robust can be sorted at a faster rate.

The table here outlines approximately how long it will take to collect 1x106 cells of different types in relation to how frequent the cell of interest occurs in the total population.

When calculating how long a sort will take, and thus how much it will cost, a flat rate charge of 1h needs to be added to cover set up costs.

How many cells do you need?

Before performing a sort you need to calculate how many cells will need to be sorted in order to collect the number of cells that you require. In calculating this you need to take a number of factors into consideration:

The percentage of your cells of interest within the total cell population. This information can be determined from the preliminary flow analysis discussed earlier. For example, if you require 1x105 cells of a particular type and this cell type is present at 10% of your total cells then you will need to sort at least 1x106 cells. If it is only present at 1% then a least 1x107 cells will need to be sorted.

During sorting there is a process termed 'sample aborts' which is necessary to achieve good sort purity, but causes cells of interest to be lost. Sample aborts usually result in loss of 10% of the total cell number in well prepared samples, but can be as high as 20% in samples which contain more cell doublets, where the cell population to be sorted is low (<5%), or at higher sort speeds.

Cells will be lost during the labelling/staining process. What you start off with is not what you will have after labelling, no matter how careful you are. Assume that at each washing step you will lose 10% of your total cells.

Once all labelling steps are complete, samples will need to be filtered immediately before sorting to remove clumps and this process can result in a lot of cell loss.

Preparation of samples

There are many factors to take into consideration during sample preparation and will depend a lot on the type of cells that you have. A detailed sample preparation/labelling procedure can be planned during discussion with the operator, but a number of more important points to consider are as follows:

It is essential that, as much as possible, a single cell suspension is maintained at all times during sample preparation. This can be a problem when working with particularly 'sticky' cells (e.g. Macrophage) or cells that normally grow attached to a substrate (e.g. Many cell lines).

You will probably already know how best this can be achieved for your particular cells, but it is well worth trying to optimise the conditions. Re-suspending cells in Ca2+/Mg2+ free media containing 0.1mm EDTA during sample preparation can help prevent the formation of clumps. However, please check that this won't adversely affect your sample or the labelling regime used.

Immediately before it is brought for sorting the sample will need to be filtered through a special filter of 50μm pore size. This will ensure the removal of any larger clumps before the sample is introduced into the cell sorter. If this is not performed, and your sample does contain clumps, it will block the machine and it will not be possible to sort the sample.

Samples should be provided at a total cell concentration of approximately 1x107 cells/ml after all labelling and filtration steps, or in a minimum volume of 300μl if only low cell numbers are available.