Proper sample preparation is absolutely essential for obtaining good purity and yields. After the final wash, cells should be re-suspended at concentrations of 20-30 million (for primary cells) or 5-10 million (for cell lines) per mL in FACS Tubes (see below). The media can be any buffered isotonic salt solution (such as PBS) but we recommend Hanks Balanced Salt Solution (HBSS) calcium and magnesium free containing 10 mM HEPES, pH 7.2).
Most cells benefit from having some protein in the media, such as 1-2% FBS, calf serum or BSA. Higher serum concentrations can lead to cell aggregation and clogging. For cell line samples, we recommend also adding 1mM EDTA. We recommend using buffers that do not contain phenol red because phenol red causes an increase in background fluorescence, and reduces sensitivity. This effect is particularly bad for dim green signals.
For sticky cells: Increase the concentration of the EDTA to 5mM and use FBS that has been dialyzed against Ca/Mg++ free PBS or 0.5% BSA. Some cell types, however, can be sensitive to high concentrations of EDTA.
For adherent cells: To detach adherent cells, trypsin (or other detachment buffer) is often quenched with media or buffer containing serum. This reintroduces cations that can promote aggregation. Use FBS that has been dialyzed against Ca/Mg++ free PBS or increase the EDTA concentration to 5mM or higher (Some cell types, however, can be sensitive to high concentrations of EDTA.). Accutase and Accumax are cell detachment solutions that can be used to generate single cell suspensions.
NOTE: Accutase can alter some cell surface epitopes; the effect will need to be determined empirically for the epitopes being evaluated.
For samples with a high percentage of dead cells: DNA from dead cells can cause cell clumping. DNAse I in the presence of magnesium chloride will help reduce cellular aggregation.
- Treat cells for 15 to 30 minutes in a solution of 100 µg/mL DNAse and 5 mM MgCl2 in HBSS at room temperature.
- Wash the cells once in HBSS with 5 mM MgCl2.
- Gently resuspend the cells in HBSS with 5 mM MgCl2 and add 25-50 µg/mL DNAse (as a maintenance dose) prior to and during the sort.
Note: DNAse I requires at least 1 mM magnesium to work effectively, although 5mM is optimal. It is important to minimize the presence of dead cells during this procedure, since actin released from dead cells irreversibly inhibits DNAse I.