Q 1: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations?

A: a) When very high purity (95%-100%) of the target population is required.

b) For separation of populations that have a low density of receptors on their surface.

c) For enrichment of populations on the basis of surface receptor density.

d) For separations on the basis of multicolor staining.

e) For separations on the basis of internal staining e.g of DNA or of internal antigens.

Q 2: Will my cells be harmed by the sorting process?

A: Generally, the cells will be not harmed through the process itself as long as they are maintained continually at a temperature, pH, and in media that is most suited to them.

Q 3: How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells?

A: 1 X 106 = 10 % target population x 50% recovery x 20 X 106 starting cell number. 50% recovery is a reasonable number, but the actual percentage of cells that are recovered with a particular sort depend on a multitude of factors:

a) Cell death that occurs pre- and post sort and loss through adherence of cells to tube walls.

b) Sort rate; the higher the sort rate the lower the recovery.

c) Precision of sort set up of the flow cytometer.

d) If it is an enrichment sort or a purity sort. Enrichment sorts have higher recovery than purity sorts.

Q 4: Are there ways to improve sort recovery?

A: Yes.

a) Use polypropylene tubes for cell processing and sorting.

b) Count cells immediately prior to sorting (after all washes).

c) Use blank Hank’s instead of PBS as sheath fluid.

d) Use polypropylene collection tubes and fill them > 1/3 with media that contains 20% serum.

e) Find optimal temperature for sort (4C, 15C, RT).

f) Invert tubes every hour during the sort.

g) Process collection tubes immediately after they are filled up or the sort is finished.

h) Spin collection tubes ~ 10 min.

Q 5: Do I have to use the monoclonal antibodies for staining of the surface antigens at the same cell to reagent ratio as for small cell numbers?

A: Our experience suggests that incubating 20 to 30 million cells in a volume of 0.5 ml of buffer with a quarter to a fifth the amount of antibody that one would calculate as the correct amount through scaling up is sufficient for adequate staining.

Q 6: How long does an average sort take?

A: The average sort takes about 1 hour of set up time for the instrument, up to 15 minutes for setting sort regions for the cells, and 30 minutes of post-sort analysis. Sorting of 20 X 106 cells takes about 2 to 4 hours. The actual time however, is highly dependent on the percentage of the desired cells in the sample as well as the purity and the number of cells that the investigator requested for the post sort assay. The set up time of the flow cytometer for a sterile sort is approximately 90 minutes.

Q 7: Is it possible to sort for positive cells and negative cells at the same time?

A: Yes, they can be collected into a left and a right sort vial. It is also possible to collect the target cells into one vial and all the remaining cells - the waste stream- into the other vial.

Q 8: What % of the theoretical number of cells can be recovered in reality and what does it depend on?

A: Generally, 50% of the theoretical number of cells can be expected to be recovered. The losses are caused by factors that have already been discussed in the answer to question 3.

Q 9: How do I assure cell viability during the sort and are there any ways of improving it?

A: Samples should be held in a rich medium that is most suitable to them. High serum concentrations are advisable because during the sort this concentration is diluted in the collection vial by sheath fluid. The collection vials are maintained at the optimal temperature and contain media for preservation of cell viability. Processing of the sorted cells as soon as possible after the completion of the sort helps to maintain cell viability.

Q 10: Is there an optimal suspension medium for the cells and an optimal cell concentration for a sort?

A: We resuspend cells at a concentration of 6-8 X 106 per ml in RPMI 1640 containing 25 milli-Molar Hepes and 2% to 10% serum because this permits to run the sort sample at a low sample pressure which improves resolution of cell clusters. However, for cell preparations which tend to clump excessively, it can be advantageous to resuspend the cells at 4-5 X 106 per ml.

Q 11: How many cells per second can be sorted on a FACStar flow cytometer?

A: Maximum event rate on the instrument is 10,000 events per second. For high purity, a rate of 2,500 events per second is typical and advisable.

Q 12: What is the maximum purity of a population that can be achieved and what does it depend on?

A: Maximum purity is 99% to 100%. 95% to 100% purity can be expected for populations that are well resolved from the unwanted cells. It is dependent on flow cytometer stability and the way sort gates are set. These gates should always be set with the investigator present to help make decisions.

Q 13: It is possible to do a sterile sort and put the sorted cells into tissue culture?

A: Yes. Whenever possible, multiple cultures should be set up from the collection vials that come off the flow cytometer. If one of the vials is contaminated the others will allow the continuation of the experiment.

Q 14: Is it possible to sort for a population that comprises less than 1% of the total or do I have to enrich for it before the sort?

A: Yes. Rare event sorts can be done but often have low purity and a low yield. Therefore, whenever possible, cells should be enriched through bulk methods or an enrichment sort.

Q 15: It is possible to exclude dead cells from the sorted population by adding propidium iodide or will it have toxic effects on the sorted population?

A: Live cells can be sorted on the basis of dead cell exclusion with low concentration propidium iodide (PI). The toxic effects of PI on the live cells have not yet been determined in our laboratory. The effects should be minimal because PI adheres only passively to the live cells and should be removable by washes. The same principle also applies to the addition of 7-aminoactinomycin D for dead cell exclusion.

Q 16: How many parameters can be used simultaneously for sort decisions on the FACStar flow cytometer?

A: The number of parameters currently available is 6; four fluorescent parameters, forward scatter and side scatter. Fluorescent area and width, and the ratio between parameters may become available as sort parameters in the future .

Q 17: How is the purity of the sorted population determined?

A: By re-analysis of the sorted population on the flow cytometer.