Flow Cytometry Laboratory - NUS Medical Sciences Cluster

Cell Sorting

Cell Sorting

Sorter operation

  • Sorters are prepared¬†

Sample tubes

  • Suspension samples may be placed in 1.5ml Eppendorf tubes, 12 X 75mm tubes, 15ml or 50ml tubes.

Sample concentration

  • Recommended cell concentration is 1X106 to 5X106 per ml of buffer/medium.
  • Beckman Coulter Moflo Astrios can run at 10 000 events per second while BD FACS Vantage runs at 3 000 events per second.
  • The nozzle size for Astrios is 100um and Vantage is 70um.

Sample filtering

  • Samples must be filtered with 60um nylon mesh (provided in the facility) to remove clumps that might plug the instrument during sorting
  • Unfiltered samples will not be allowed to sort
  • For samples that tend to aggregate over time, you may add some DNase
  • Add 0.02mg/ml DNase type IIS to all cell preparation steps, including wash steps, to eliminate free DNA from broken cells that leads to aggregation.

Collection

  • Sorted cells can be collected into
  • tubes: 1.5ml Eppendorf tubes, 12 X 75mm tubes, 15ml or 50ml tubes.
  • plates: 96-well and 384-well plates

Culture media or buffer or serum may be used in the collection tubes/plates

Tubes/ Plates Volume of collection media/buffer/serum
1.5ml Eppendorf tube 0.5ml
15ml tube 3ml
50ml tube 5ml
96-well plate 200ul
384-well plate 50ul

 

  • It is recommended to spin the plates post sorting for 30-60s at 300G to help the cells to adhere and increase the number of colonies that will grow.
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