Sorting buffer
The basic sorting buffer consists of:
1X DPBS or HBSS (without Ca or Mg)
1 mM EDTA
1 % inactivated FBS (or BSA)
Sterile filter and keep at 4°C
For cells that tend to aggregate easily, you can add up to 5 mM of EDTA (careful: too much EDTA can kill your cells!). 25 mM HEPES pH7 can also be added.
Dead cells will leak DNA in the media which will cause clumping and can potentially reduce the overall yield of the sort. If you suspect that your sample contains a lot of dead cells, treat your sample with DNase prior to the sort.
Straining your cells right before coming to the facility is recommended to prevent clumping (and clogging).
Sample concentration
In general, the cell concentration for common type of sample:
Keep in mind that cell concentration will influence the speed of the sort, but it can also affect its efficiency. The cells' size, their fragility, the quantity of debris or the proportion of the desired cells in the sample are all parameters that will help determine the ideal cell concentration to use (and the choice of configuration). This is why you need to talk with the facility manager before coming for a sort and give as much information about your cells as you can.
Collecting samples
Cells can be sorted onto slides, into plates (6 to 96 wells), 5 ml or 15 ml tubes or microtubes.
The size of the collection tubes depends on the number of population you wish to sort and the number of cells you expect to get. In general use:
Because polystyrene generates a lot of static electricity, it is preferable to use polypropylene tubes for sorting. To ensures that your cells don't dry out during the sort, make sure to fill your collection tubes with 50 µl (microtubes), 500 µl (5 ml) or 1 ml (15 ml) of either FBS or sorting buffer. Because the sorted cells are in a PBS droplet, any solution in the collection tube will be very diluted. For this reason, sorting into media is not recommended for bulk sorts: it won't help the cells' recovery and in certain cases, it can even be detrimental.
For single cell sorting into plates, fill the plate with media at the volume you would normally use to plate cells.
While the sort is performed under aseptic conditions, as an additional protection, we recommend that you supplement your media with pen/strep for one or two passages whenever it is possible.
The basic sorting buffer consists of:
1X DPBS or HBSS (without Ca or Mg)
1 mM EDTA
1 % inactivated FBS (or BSA)
Sterile filter and keep at 4°C
For cells that tend to aggregate easily, you can add up to 5 mM of EDTA (careful: too much EDTA can kill your cells!). 25 mM HEPES pH7 can also be added.
Dead cells will leak DNA in the media which will cause clumping and can potentially reduce the overall yield of the sort. If you suspect that your sample contains a lot of dead cells, treat your sample with DNase prior to the sort.
Straining your cells right before coming to the facility is recommended to prevent clumping (and clogging).
Sample concentration
In general, the cell concentration for common type of sample:
- Inactivated PBMCs, splenocytes, thymocytes, whole blood, bone marrow: 20 million cells/ml
- Activated PBMCs, splenocytes, thymocytes, B and T cell lines: 10 million cells/ml
- Primary cells, embryonic stem cells, DCs, transfected cells: 8 million cells/ml
- Epithelial cells, adherent cells, delicate cells, recently transfected cells, large cells: 3-5 million cells/ml
- Cells to sort in plate as single cells: 1 million cells/ml
Keep in mind that cell concentration will influence the speed of the sort, but it can also affect its efficiency. The cells' size, their fragility, the quantity of debris or the proportion of the desired cells in the sample are all parameters that will help determine the ideal cell concentration to use (and the choice of configuration). This is why you need to talk with the facility manager before coming for a sort and give as much information about your cells as you can.
Collecting samples
Cells can be sorted onto slides, into plates (6 to 96 wells), 5 ml or 15 ml tubes or microtubes.
The size of the collection tubes depends on the number of population you wish to sort and the number of cells you expect to get. In general use:
- 15 ml tubes if you want to sort 1 or 2 populations and expect to retrieve more than 1 million cells
- 5 ml tubes if you want to sort up to 4 populations or expect to retrieve between 0.2 and 2 million cells
- Microtubes if you want to sort up to 4 populations but don't expect to retrieve more than 200 000 cells
Because polystyrene generates a lot of static electricity, it is preferable to use polypropylene tubes for sorting. To ensures that your cells don't dry out during the sort, make sure to fill your collection tubes with 50 µl (microtubes), 500 µl (5 ml) or 1 ml (15 ml) of either FBS or sorting buffer. Because the sorted cells are in a PBS droplet, any solution in the collection tube will be very diluted. For this reason, sorting into media is not recommended for bulk sorts: it won't help the cells' recovery and in certain cases, it can even be detrimental.
For single cell sorting into plates, fill the plate with media at the volume you would normally use to plate cells.
While the sort is performed under aseptic conditions, as an additional protection, we recommend that you supplement your media with pen/strep for one or two passages whenever it is possible.
