We have three cell sorters across our two sites. On these pages you'll find support for using them.
We have three cell sorters at the Facility. You'll find information about them on our equipment pages.
Bringing & Collecting Cells
Find information on bringing and collecting cells at the Facility.
Bringing your cells
Number of cells to bringNumber of cells to bring
The number of cells you need to bring depends on:
- How many cells you require for downstream work.
- How rare your cell population is within your pre-sorted sample.
If you know roughly these two variables you can make an estimate for how many cells you need to bring.
Calculating your cell estimateCalculating your cell estimate
We recommend that you bring around 20% more cells than your estimate. This is to allow for inaccuracies such as:
- a percentage of any pre-sorted sample will be non-viable (not desirable for sorting)
- any cell counting method has a level of inaccuracy
- finding that you have fewer cells in your pre-sorted sample than you had thought
You should also decide whether you are counting your cells before or after staining and washing. Numbers will differ based upon when you count.
Our sorters all give a cell count of the sorted cells. This number is at best 85% accurate.
If your downstream application requires a minimum number of cells, please allow a 15-20% margin of error extra within the sorted population.
You want 100,000 total cells. Your target population is 1% of the total cell population.
100,000 x 100 = 10,000,000 cells + 20% extra = 12,000,000 total cells needed.
Buffer and concentrationBuffer and concentration
We recommend that you bring your cells in FACS tubes in a buffer the cells are happiest in.
Decrease the protein concentration of the buffer to a maximum of 2%.
We recommend a cell concentration of no more than 2x107 per ml.
Bring an extra buffer to the sort as we may need to dilute your cell sample.
Filtering your cellsFiltering your cells
If your cells are adherent or there is a large amount of dead and dying cells in the population you may find that cell aggregates form (clumping).
This can easily block the sorter and delay your sort significantly.
To ensure this doesn’t happen we may ask you to filter your cells to remove aggregates.
We sell 30um filters at the facility. These are not sold sterile but you can also bring your own.
Rule of thumb is that the filter size should be around half that of the sort nozzle used.
For example, cells that are to be sorted through a 70 um nozzle = 30 um filter. Cells through a 100 um nozzle=50 um filter.
It is pointless to filter cells through a 100 um filter then expect them to pass freely though a nozzle of the same diameter.
Tubes, plates, microscopic slidesTubes, plates, microscopic slides
We have the capacity to sort into:
- tubes (eppendorfs, FACS Tubes, 15ml Falcons)
- plates (384 wells to 6 wells)
- microscope slides
Specify what you are planning to collect in before the sort is set up to avoid any delays.
The maximum number of simultaneous sorted populations are:
- Eppendorf -4 (6 on Astrios)
- FACS Tube -4 (6 on Astrios)
- 15ml Tube -2
- Microscope slide -3
- Any plate format -1
We recommend that you collect your cells into a buffer. This avoids stressing the sorted sample.
The larger the volume of collection buffer the lower the stress on the cell. But, this will mean you can collect fewer cells per tube.
Collect your cells into neat FBS/FCS or high concentration FBS/FCS cellular media. This can improve the survival of your sorted sample.
Find information on the sorting process at the Facility.
Number of cells we can sortNumber of cells we can sort
The number of cells we can sort in an hour depends on the size of your cells and therefore the size of the sort nozzle we can use.
The smaller the sort nozzle the faster we can sort. 70um is the smallest nozzle. We then have 85, 100 and 130um nozzles.
|Fusion, Aria II and Aria III||Astrios|
|70um||30 million/hour (60PSI)||40-50 million/hour (60PSI)|
|100um||15 million/hour (20PSI)||20-30 million/hour (30PSI)|
Nozzle choiceNozzle choice
The nozzle choice is dependent upon the size of the cells in your sample.
As a rule the nozzle should be 4x bigger than the largest cells in your sample.
As a general rule:
- PBMCs (8-12um) require the 70 or 85um nozzle
- cell lines and bigger primary cells (12-20) require the 100um nozzle
- anything larger requires the 130um nozzle
Be aware that the smaller nozzles have higher pressures. Some smaller cells do not like this pressure. They can be sorted on larger nozzles with a resulting drop in rate.
Sterility processSterility process
Our sorters are all sampled for sterility every week. They're cleaned thoroughly on a regular basis.
We also shut our sorters down with 70% ethanol every night to avoid any contamination and to preserve the sterility.
Post-sort culturing is common practice and infections are rare but can occur.
If contamination occurs
If your sorted cells are infected, let us know. We will identify if it was the sorter or some other source of contamination.
To decrease the risk of contamination, prepare your sample in tissue culture facilities with proper aseptic technique where required.
Other equipment in the core facility (such as our lyse wash assistant) is not screened for contamination. This can be a source of contamination if used before sorting.
Temperature for sortingTemperature for sorting
We can adjust the temperature of your sorted sample in two locations:
- Your pre-sorted tube can be controlled at 4, 20, 37, and 42oC within the instrument.
- Your collection tubes/plates/slides can be controlled to any temperature between 5oC and 42oC.
The temperature you sort at is dependent on your cells and your downstream assay.
If you want to culture your cells after sorting it may be preferable to sort at 37oC.
For other protein or nucleic acid work, cooler temperatures may be preferable. This can preserve the integrity of your sample in the collection device.
If in doubt as always it is best to ask a member of the FCCF team.
Sort masksSort masks
The 'sort masks' are a way for us to influence the sort decision made by the sorter.
In broad terms the sorter has two factors. These work in opposition to one another: purity and recovery (yield).
To obtain every single target cell from a heterogeneous population you lose the purity of the sorted population.
Equally to sort a population with high purity you will sacrifice the yield of the population.
We strive to strike a balance between purity and recovery. This can be done by altering the 'droplet envelope' ('sort mask').
For example, for a single cell (single droplet) sort, the sort mask is set to be the highest purity at the loss of recovery.
FCCF staff will discuss your needs and determine the best sort mask for your experiments.