Sorting

Flow Cytometry

Fluorescence Activated Cell Sorting

This is the process by which cells are physically separated from a sample based on their different florescence and scatter properties. When a cell is excited by the laser and meets the criteria defined by the user as a cell of interest, the stream is charged. Depending on the number of cell populations being sorted, individual cells are deflected to go into the appropriate collection device.

Up to 4 non-overlapping populations of cells can be sorted simultaneously, with the unselected cells going into a waste container. Cells can be sorted into 12x75mm tubes, 15-ml tubes, 6/12/24/96 well plates, or onto a microscope slide.

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The process can be done under aseptic or non-aseptic conditions. Once cells are sorted, they can be used for further analysis or for re-culture. Live cells and fixed cells can be sorted. Live cells should be suspended in whatever media keeps them the happiest. Complete media with pH indicator and serum supplements in no way interferes with the ability to detect fluorescence.

Cell concentration can be as high as 5x10⁶ cells per ml, and is preferred to ensure optimal control of the sample pressure during the sort. Dilute samples can be used; however, these samples take longer to run. Fill the tube containing your sample to no more than 75% capacity. Samples must be in Falcon 2058 tubes (5-ml polystyrene round-bottom tubes, 12x75mm).

For the most efficient sorting, filter all samples (normally we use a 35 micron filter cap tube). A clog that may develop in the nozzle during routine analysis presents no problem, but if a clog develops during a sort, it can ruin an otherwise perfect sort by causing unwanted cells to be deposited into the collection tube.

The use of antibiotics is recommended for sterile sorts. The addition of 300 Units/ml of penicillin G and 0.3 µm/ml streptomycin sulfate has worked well. Also, 100 µm/ml gentamycin can be added for extra protection along with the pen/strep. It is not recommended that gentamycin alone be used as a sole source of antibiotics. Also, G418 can be added if the cells are resistant to neomycin.

Collection tubes should be set up a day in advance, and should be completely filled with media, BSA, or 100% FCS and stored at 4°C to limit the adherence of the charged cell droplets to the sides of the tube during the sort. Most of the collection tube liquid will be removed immediately prior to the sort. Bring extra collection tubes in case the primary tube gets contaminated or mis-sorted.

Sorting is stressful to the cells and we need to give the sorted cells every chance to recover and survive after the sort.

The time it takes to sort the cells depends on the percentage of the total population you are interested in, and the purity of the initial sample (free of clumps and other particulate material). A typical throughput for the cell sorter can be between 8,000 and 12,000 cells per second. For example, under optimal conditions, if the population you were interested in was 10% of the total, you could sort 800 cells per second. At this rate it would take 30 minutes to recover 1x10⁶ cells.

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