Image - Barts and The London logo and link to home page Image - divider Image - divider
 
  Institute of Cell and Molecular Science
Flow Cytometry Core Facility equipment, images used with permission by BD
  Flow Cytometry Core Facility
  link Home link Instruments available link Flow cytometry link Uses of flow cytometry link Flow sorting link Links link Contacts
 
Sort times

Sorting with the BD FACSAria

With the FACSAria, particle position is known to within 1/32 of a drop, and with extreme stability of the sort set-up, the maximum sort envelope is 2 drops, when the "Recovery Mask" is set to its maximum value of 32.

Running the FACSAria sorting lymphocytes in High Sort mode at 70 psi and drop-drive frequency of 90 KHz, it is perfectly feasible to sort with high purity at analysis rates of 20,000 per second. [Sorting for yield rather than purity can be done significantly faster still.] The actual number of target cells that are sorted out of the mixture will be lower, proportional largely to their frequency, so for a 10% target population, collecting 2000 target cells per second is a reasonable number.
Sorting cells >10um can be achieved by use of the 100um diameter nozzle at 20psi with sort rates of 10,000 cells per second are optimum at 30KHz. For very large cells >40um or fused cells a 130um diameter nozzle at 12psi can sort cells at 5,000 per second.

Sorting at conventional speeds

As useful guide, a flow cytometer operating at 3000 events per second processes about 107 events per hour not taking any abort decisions into account. For typical 'biochemical' experiments large numbers of cells are required and flow sorting has its limitations in these areas because it is too time consuming. The arrival of PCR-based techniques has restimulated interest in flow sorting, as experimentation with individual and low numbers of cells has become a practical reality.

Sorting sub-populations of less than 5%

Low frequency sub-populations are sorted at only a few events per second. These need careful collection; being of like charge, the droplets repel each other and have a tendency to evaporate before coalescing, collecting into a tube already containing liquid is essential. Recoveries tend to fall dramatically as smaller sub-populations are investigated.

A useful strategy when attempting to isolate significant numbers of cells from sub-populations comprising less than 5% of the whole, is to adopt a two stage procedure. After a preliminary estimate of the frequency of the sub-population, the flow rate is adjusted to give, on average, one cell per droplet.. The required analytical rate would now be that of the drop drive frequency i.e. up to 50,000 per second which is well above the capabilities of conventional flow cytometers and sorters. However, if the required cells are fluorescently labelled it is then possible to trigger the machine such that it only detects positive cells at a frequency it can then process. The unwanted cells are then treated in the same way as debris below a conventional scatter threshold, that is to say ignored. If three droplets are charged and sorted we would expect a 33% purity and high recovery; the sort decisions will not be aborted as the unwanted cells are not detected. This strategy becomes more effective as the sorted population gets smaller.

The table below illustrates some of the sort times required for the collection of cells from different starting populations.

Sort times given % positive in starting population
Assuming flow rate of 3000 cells per second 107 cells per hour
No. of cells requested 0.1% 1% 5% 10% 20%
1000 5.5mins 33sec 6.5secs 3.3secs 17.secs
10,000 55mins 5.5mins 1.1mins 33secs 17secs
100,000 9.8hrs 55mins 11mins 5.5mins 2.8mins
1,000,000 3.8days 9.2hrs 1.8hrs 55mins 28mins
10,000,000 38days 3.8days 18hrs 9.2hrs 4.6hrs

 

Flow sorting and magnetic cell sorting (MACS)

Clearly as shown in the table above there are situations in which flow sorting is not practicable to obtain the required number of cells. In such situations MACS should be used. The MACS microbeads are superparamagnetic particles that are coupled to highly specific monoclonal antibodies. They are used to magnetically label the target cell population. They are approximately 50 nm on size, biodegradable and do not affect the cells. Labelled cells are placed in a column within a magnet. By rinsing the column all unlabelled cells are washed from the column leaving the labelled cell fraction. By removing the column from the magnet the labelled cells can be recovered. The entire procedure can be performed in less than 30 minutes and the cells may immediately be used for experimentation.

 
Top
 
by Gary Warnes. © Queen Mary, University of London 2007
Institute of Cell and Molecular Science, The Blizard Building, Barts and The London School of Medicine and Dentistry, 4 Newark Street, London E1 2AT, UK Tel: +44 (0)20 7882 2483, Fax: +44 (0)20 7882 2200