Buffer design for both your pre-sort sample and post-sort sample collection
is very important for an optimized sort. In addition to the following basic
recipe other modifications are included that may be germane to your
Culture media is not a good sort buffer in that the calcium chloride in most culture medias will precipitate as a calcium phosphate with the phosphate present in the sorter sheath fluid.
Basic Sorting Buffer
- 100ml 10x Sterile PBS
- 1% BSA (10g BSA)
- 2mM EDTA (4ml 0.5M EDTA)
- 900ml dd H2O
- Sterile Filter
Increase the EDTA to 5mM and use FBS that has been dialyzed against Ca/Mg++
free PBS. Activated cells tend to become clumpy. The addition of the EDTA
acts to chelate the sample, reducing cation dependent cell adhesion.
Samples with Dead Cells: A large number of dead cells in the preparation will lead to an increase in the amount of soluble DNA in the solution which will coat cells, leading to profound clumping. Adding 10 U/ml DNAase II can help reduce this.