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===== What should I expect when sorting? =====

==== Do I need to operate the equipment by myself? ====

We will train you to use the analyzers on your own. The sorters can only be operated by [[about|Flow Cytometry Facility staff]]. You can book time with us and we will sort for you. But if you want to learn cell sorting, we will teach you.

==== How are my cells collected? ====

{{  ::how_many_cells.png?400|}}You have many different options for collecting your sorted cells.

  * Collect up to four different populations simultaneously into tubes of your choice, 1.5 mL, 5 mL tubes, or up to two population into 15 mL tubes.
  * Sort into any kind of plate. 6-well,12-well, 24-well,36-well, 96-well, 384-well, PCR plate, etc.

The most recommended collection buffer is your **cell culture medium with 10% FBS** or some other serum. Collection tubes should be about 1/3 full of collection media. If you want to culture your sorted cells it’s a good idea to add antibiotics Pen-strep or gentamycin, and antifungal agents to the collection media. You can also sort directly into lysis buffer (for example, buffer that contains Trizol).

We can **adjust the temperature** of the sorters to 4 ºC, 20 ºC, 37 ºC and 42 ºC, and we can also keep your cells on ice, if that is what you need.

The BD FACSAria sorters faster with a smaller nozzle at higher pressure. With the 70 µm nozzle, you can sort up to 20,000 events/second. This is a great choice for small cells that are happy in single-cell suspension, for example blood and bone marrow. Larger cells need a larger nozzle size. Your cells should be no more than one-third the size of the nozzle. Cells that are fragile or easily stressed (e.g. cultured cells) need a lower pressure sort (100 µm nozzle). Our sorters have the following options: 
  * 70 µm nozzle / 70 psi
  * 85 µm nozzle / 45 psi
  * 100 µm nozzle / 20 psi

We normally use saline (0.9% NaCl) in our sorters as sheath fluid. Each individual cell is sorted inside a tiny drop of this sheath fluid. If you are using the 70 µm nozzle (high pressure, faster sorting), you will get approximately 1 ml of sheath fluid with every 1 million sorted cells. If you are using the 100um nozzle (low pressure, slower sorting), you will get approximately 3 ml of sheath fluid with every 1 million sorted cells.

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