(Flow Cytometry Based Knowledge Portal)
Phosphate buffered saline (PBS)
Phosphate buffered saline (abbreviated as PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH(7.2). The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).
PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be ‘dried’ and immobilized to a solid surface. The thin film of water that binds to the substance prevents denaturation or other conformational changes.
PBS Buffer correct composition can significantly improve the quality of the recovered cells during sorting by tackling the concerns of viability, auto-fluorescence, and non-specific staining. Here we discuses the use of ingredient in PBS buffer :
Use Ca/Mg2+ free PBS
Absence of these ions reduces cation-dependent cell to cell adhesion and prevents clumping. A debris free single cell suspension will have lower auto-fluorescence and flow smoothly through the nozzle.
FCS (1-10%) or BSA (0.1-1%)
Serum proteins protect cells from apoptosis, prevent non-specific staining and also prevent cells sticking to the tube. However, a high concentration of serum can contribute to auto-fluorescence, therefore the optimum serum level should be pilot tested beforehand.
EDTA (0.5-5 mM)
EDTA also prevents cation based cell to cell adhesion and so should be included in the buffer if dealing with sticky and adherent cells like macrophages. However, the concentration of EDTA should be titrated first as high levels can be cytotoxic and/or cytocidal.
DNase I (25-50µg/ml)
Dead cells release their contents including DNA into the medium which can lead to cell clumping and related problems. If the sample has a high percentage of dead cells – ideally, the dead cells should be removed, but in case that is not feasible due to low cell numbers – it is advisable to add DNaseI into the buffer.
Sodium azide (0.1-1%)
Sodium azide – at suitable low concentrations – checks bacterial contamination, prevents photo-bleaching of fluorchromes and blocks antibody shedding. As with the EDTA, the optimum concentration should be pilot tested to avoid cell toxicity and death.