- Sorters are prepared
- Suspension samples may be placed in 1.5ml Eppendorf tubes, 12 X 75mm tubes, 15ml or 50ml tubes.
- 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.
- 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.
- 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|
- 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.