Question

What buffer should cells be prepared in?

Answer

• In general, any physiological buffer can be used, but it is essential to avoid cell clumps (aggregates).
• For prokaryotic cells: PBS (phosphate buffered saline).
• For eukaryotic cells: PBS or HBSS without Ca²⁺/Mg²⁺ and with ≤2% FBS or BSA. Standard media for eukaryotic cultures contain Ca²⁺, Mg²⁺, and serum proteins, which promote aggregation. Many cells benefit from the presence of proteins in the buffer (e.g., 2% FBS or BSA), but concentrations >5% can lead to aggregation and clogs.
• For cell lines and adherent cells, the addition of 0.5 mM EDTA is recommended, which can be increased to 5 mM for highly adhesive cells.
• In suspensions with a high percentage of dead cells, free DNA can coat the cells and cause severe clumping: adding DNase II (20–100 μg/mL; 10 U/mL) may help.
• For cells sensitive to pH, adding HEPES (10–25 mM) is advisable to buffer pH fluctuations caused by high pressure.
• The addition of antibiotics is recommended for sterile sorting conditions.