Fluorescence activated cell sorting allows analysis and physical sorting of individual cell-types or clones from a mixture of cells, into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent labelling of each cell type.
Cell sorts can be processed using a range of collection tubes and plates from 15ml Falcon tubes to 384-well plates that contain the specific collection media necessary for the down-stream processing for the sample.
BD FACS ARIA II parameters
Up to twelve fluorescent parameters and two physical parameters (FSC - size, SSC - granularity) can simultaneously be analysed and sorted on our instrument. To check the configuration of our FACS ARIA II Cell sorter, please see the following documents:
When designing your multi-parameter fluorescent panel, we strongly advise that you read our multi-colour analysis page. Please also refer to the fluorochrome table specific to our Aria setup to assist you.
How to prepare your cells
Correct preparation of your sample for sorting is the single most important factor in getting the best results. Samples should be a single cell suspension and filtered. We suggest a 40um filter (BD Cat No. 340632 or Falcon Cat No 352340). Unfiltered samples cannot be run.
The exact sample perpetration will depend on the specifics of your experiment, however, we can suggest the following:
- The sort buffer should be PBS + 1% FCS
- You will need at least 2.5 times the final number of cells you wish to sort:
- Too much FCS and the sample will froth and introduce contamination
- Not enough FCS and the cells may die
- EDTA may be added to reduce clumping
- Attempt to remove/reduce cellular debris and dead cells:
- Healthy cells sort and recover quicker than a mixture of live and dead cells
- Debris can slow down a sort and reduce yield
The sort compromise
The sort compromise is a balance between the yield (number of cells recovered), purity, and time taken to sort.
Every sort needs a negative control.
Other controls needed depends on specific experimental design. Ideally, there should be single colour positive controls and Fluorescence Minus One (FMO) controls to initially set up experiment parameters.
How to book the FACSAria II
Please see our Registration and pricing page for details
Sterile sorting conditions
Cell sorting is aseptic, not sterile. We perform regular cleaning regimes. You may request for the equipment to be specially prepared for aseptic sorting (PAS). This takes about one hour that needs to be booked immediately prior to the sort.
What can you sort into?
We are currently able to sort into:
- 96-, 48-, 24-, 6-welled plates (384-well plates by special arrangement)
- 0.5ml PCR tubes
- 1.5ml, 2.0ml microcentrifuge tubes
- 15ml Falcon tubes two-wayy sort only)
There is a range of nozzle sizes that can be used to increase efficiency of cell sorting (see below).
|Nozzle size (um)||Sheath pressure||Frequency (KHz)||Cell type|
|70||70 psi||90||Non-activated T cells, B cells, platelets, bacteria, yeast|
|85||45 psi||47||Activated T cells, plasma cells, iNKT cells, NK cells, monocytes, mDC, pDC, stem cells|
|100||20 psi||20||Neurons, macrophages, stem cells, cell lines|
|130||10 psi||10||Any problematic cell types using above settings|