FACStar Sorting Advice

Sorting is an art, a skill, and a science. It is, at all three of these levels, a working partnership between the research user and the flow operator. There are certain theoretical limitations that a user needs to understand about the technique itself and there are also certain things that a user can do to maximize her/his chances of sorting the required numbers of desired cells -- in good condition for further manipulation.

THEORETICAL LIMITATIONS

CELL NUMBERS: Although flow sorting may seem magical, it cannot create life: You will never end up with more cells than you started out with. If your initial sample has a million cells and you want to sort 1% of that population, then you cannot get any more than 10,000 cells at the end of the sorting procedure. In fact, there are certain built in "abort" conditions, so that your theoretical maximum could be only 50-80% of this value. Abort rates rise when sorting at high speeds and also when sorting relatively rare cell populations. The following graphs give an example of sort rate and recovery when sorting a 1% population (with a 1.5 drop envelope and a drop drive frequency of 62,000 drops per second). When your original cell suspension is at a high concentration, you will sort at a fast speed; at fast speeds, the sort will go more quickly, but more cells will be lost (low recovery). You need to decide on the optimal flow rate, based on how precious your cells are.


Use the lab spread sheet to make these calculations for your own sorting conditions. You must make sure that you start out with enough cells to cover your requirements and some extra to cover abort losses (experienced sorters usually try to start out with at least twice the number of cells that they need -- just to be safe).

TIME: The flow cytometer sorts best when it is processing cells at a rate of about 2000 — 20,000 cells per second. In order to give this rate, the unsorted cells should be at a concentration of about 2-20 million per ml. When cells flow at a rate of 2000 per second, the total number of your original particles that will be processed each hour is 7 million; if your desired particles are 1% of the total, then you will sort those particles at a rate of about 70,000 per hour. If, however, your desired particles are 50% of the total, then they will sort at about 3.5 million per hour. This information may help you to plan your time. It may also help you to plan your budget -- and may convince you to do anything you can to enrich your cell population before you bring your sample to the flow lab. Aside from financial considerations, it is also true that the less time you spend sorting, the more viable will be your sorted cells. The following graph indicates the predicted time for a sort of a million cells from a population in which they originally represented 1% of the total.


HINTS

RECOVERY/VIABILITY: After an effective sort, it is a pity to lose precious cells as a result of careless post-sort handling. Bearing in mind the theoretical limits mentioned above, cell recovery and viability after the sort event can be maximized by some simple procedures. Because cells will get diluted into cytometer sheath fluid (usually PBS) during sorting., it is important to sort cells into tubes that contain a small volume of appropriate medium in order to keep the cells happy and viable after they have traveled through the laser beam and been deflected to the left or right. Different types of cells have different preferences. You need to decide on the appropriate serum or medium into which you want to sort your cells. It is also possible to change the sheath fluid if your cells do not like PBS. It is up to the user to consider the cells' preferences and discuss these with the sorting operator in advance.

For blood cells, it is best to coat the receiving tubes with serum and then to sort into a volume of about 0.5 ml of 50% serum in buffer. Another factor to remember is that the cells to be sorted (and the cells that have already been sorted) are not being kept under carbon dioxide. Therefore use a HEPES or similar buffer, but avoid bicarbonate buffer as it will not maintain an appropriate pH for long.

If you are sorting into tubes, you should therefore bring to the flow lab Falcon tubes that have been coated with serum and that contain about 0.5 ml of serum/medium -- and bring enough of those tubes to receive all your sorted cells. Bring one 3 ml tube containing 0.5 ml of serum/medium for every 500,000 cells you plan to collect (and three extra tubes just to cover any problems that may occur). If you are sorting into the wells of a plate, put 100ml of serum/ medium into each well.

As mentioned above, a final factor is time: the more you can do to pre-enrich cells before sorting, the faster they will be sorted and the happier they will be.

STERILITY: Although the flow sorter is a cumbersome beast, it can be sterilized to provide the user with sorted cells for long term culture. The sort operator needs to be informed in advance about a requirement for sterility since flow lines, tubes, and filters all need to be specially treated. Because there is no such thing as absolute sterility (and effective sterility will vary depending on the antibiotics present in your culture medium), if you intend to grow sorted cells for a long time without much antibiotic cover, you may want to use a short test sort at some time before the big experiment to check flow sterility under your own culture conditions.

PURITY: Cell sorting will, routinely, produce populations that are 95-99% pure for the desired cells. Apparent lack of purity can result from various reasons that have nothing to do with the effectiveness of the sorting procedure. For example, purity will be less if the sorted cells are not a clearly discrete population and overlap with unwanted cells. Also, clumps of cells that separate after sorting may make it appear that a sorted fluorescent population is contaminated with non-fluorescent cells. Loss of viability after sorting may result in cells with different scatter properties from those of the original cells. Sorting cells away from platelets/debris is often not effective because platelets and debris stick to sorted cells and fall off after the sort. Capping of surface markers after the sorting procedure will often result in sorted cells that are somewhat less fluorescent than the original selected population. But, with these limitations, we can expect to take cells that are, for example, less than1% in an original sample, and end up with about 98% purity. However, you shouldn’t take this for-granted. At the end of every sort, you should run an aliquot of the sorted cells through the cytometer in order to check their purity in comparison with the original, pre-sort sample.

CONTROLS: Cell sorting is a further elaboration of the flow technique used for cell analysis. Controls are, therefore, just as important for sorting as for non-sorting flow cytometry. If you want to sort fluorescent cells, then you need to run control or unstained cells first in order to see where to "draw the line" between the fluorescent and non-fluorescent populations. What this means is that you will have to bring a tube of about 300,000 unstained cells (as well as tubes of single-stained cells to set the compensation in multi-color protocols) in order to assist the sorting operator in setting the cytometer to make its sort decisions. These control cells should, of course, be sterile if you are doing a sterile sort (so that they will not contaminate the clean cytometer).

CHEAT SHEET

1. Discuss the sort well in advance with the sort operator. In order to prepare for the sort, the operator will need to know

what kind of cells will be sorted,

how many cells will be brought to the cytometer,

what stains will be used,

what proportion your desired cells are of the original population,

how many cells will be required,

whether you want to sort into tubes or into a 96-well plate,

whether the cells need to be kept cold during sorting,

what sheath fluid will keep your cells happy,

and whether or not the sorter needs to be sterilized.

2. Bring to the flow lab twice the number of unsorted cells that you calculate are needed to provide you with enough of your sorted population. Do everything you can to enrich your cells before you get to the flow lab. Bring an extra tube with about 300,000 unstained (control) cells if you want to sort a population based on fluorescence. Bring tubes with single-stained cells to set the compensation in multi-color protocols.

3. For sorting, your cells should be made up to a concentration of about 2-20 million per ml. Plan your time by remembering that your unsorted cells will be flowing through the cytometer at a rate of about 7-70 million per hour. Faster rates provide quicker sorting, but decrease the % recovery. It also helps the operator if some extra medium is available in a separate container so that cells can be diluted if the initial flow rate is too high.

4. Bring serum-coated Falcon tubes (or a plate) containing medium/serum into which the cells will be sorted. If using tubes, bring one tube for every 500,000 sorted cells (plus three extra in case of problems).

5. Re-run a few of the sorted cells at the end of the sort to check their purity. The sort operator would also appreciate feedback about how many sorted cells you count and about their viability (and sterility, if relevant).