Our customers are responsible for the adequate preparation of cells to be sorted(incl. staining) as well as for adequate negative controls and single stained controls as necessary. However, we would be happy to offer every assistance necessary for planning and successful preparation.
- All cells need to be filtered through a nylon mesh prior to sorting 40µm for sorts with the 70µm Nozzle, 70µm for sorts with 100µm Nozzle and above). It proved advantageous to resuspend cells that tend to aggregate in EDTA- containing buffer (e.g. PBS, Ca/Mg free, 0,5%BSA with 1-2mM EDTA). It is further recommended to store cells on ice prior to sorting.
- In samples with low viability, aggregation may occur due to the release of DNA. This can be mitigated by supplementation with 10 U/ml DNAse in the presence of 1-5 mM MgCl. Please do not use buffer with EDTA if using DNase!
- The concentration of cells for sorting should be in the range from
5 x 106 cells/ml (e.g. for large adherent cells) to
50 x 106 cells/mlp (e.g. for lymphocytes)
depending on total cell count, cell size and tendency to aggregate. However, the initial volume should not be less than 250µl.
- The cell suspension can be applied for sorting from 1,5 ml Eppendorf-Cups, 5 ml (12x75 mm) Tubes, or 15 ml Tubes (additionally 50ml tubes for MoFlo und MoFlo XDP).
- It is highly recommended that tubes to be used as sample tubes be made of polystyrene (clear), while those for sample collection be made of polypropylene (opaque), because of their differing electrostatic properties.
- Collection tubes for the target populations supplied with the recommended medium for these cells need to be provided by the customer, e.g.
- Fetal bovine serum only,
- your own culture media with antibiotics
Note: A conclusive reanalysis of your cells is only possible with filtered (0.2 µm) collection medium (especially when containing FCS!)
- Since the sorting procedure is putting cells under considerable stress and due to the dilution of medium through the PBS- containing sorting drops generated by the machine, the recommended concentration of serum and proteins in the collection tubes needs to be significantly higher compared to cell cultivation (Dilution with PBS through sorting: 1nl per sorted cell for 70um nozzle, 3nl for 100um nozzle).
- 1,5 ml Eppendorf-Cups, 5 ml (12x75 mm) tubes and 15 ml tubes (NOT for sorting more than two populations simultaneously!) as well as cell culture plates (6 - 384 wells) can equally serve as collection tubes for sorted cells.
Please note: Only 5 ml and 15 ml can be used as collection tubes for sorting of infectious samples or genetically modified organisms of safety level S2.