Flow cytometry is a powerful laboratory technique used to analyze and sort cells based on their physical characteristics and fluorescent markers. The procedure requires cell samples to be suspended in a specialized liquid called FACS buffer, an abbreviation for Fluorescence-Activated Cell Sorting buffer. This precisely formulated solution serves as the cell’s temporary environment during the staining, washing, and analysis steps. It keeps cells stable and intact from the moment they are harvested until they pass through the instrument’s laser beam, ensuring accurate data.
Maintaining Cell Viability and Structure
The primary function of the FACS buffer is to maintain the physiological integrity of the cells by controlling the solution’s properties. The buffer must be isotonic, meaning it has the same osmotic pressure as the cell interior. If the buffer is hypotonic (less concentrated), cells swell and burst (lysis). Conversely, a hypertonic buffer causes cells to shrink (crenation). Both extremes damage cell membranes and distort light scattering properties, leading to incorrect measurements.
The buffer also stabilizes the hydrogen ion concentration, or pH, typically maintaining a neutral environment around 7.4. A stable pH ensures cellular proteins and antigens retain their correct shape and function throughout the procedure.
Key Ingredients and Functional Roles
The foundation of a standard FACS buffer is a balanced salt solution, such as Phosphate-Buffered Saline (PBS). PBS provides the necessary sodium and chloride ions to maintain isotonic balance and includes phosphate salts that serve as the primary buffering agent to control the pH.
The solution is supplemented with a protein component, usually Bovine Serum Albumin (BSA) at a concentration often ranging from 0.1% to 1.0%, or Fetal Bovine Serum (FBS) at 1% to 10%. This added protein prevents the non-specific binding of fluorescently labeled antibodies to the cell surface. By occupying random binding sites, the protein reduces background fluorescence, allowing accurate measurement of only the specific binding of the target antibody.
Another frequent component is Sodium Azide (\(\text{NaN}_3\)), typically added at a low concentration of 0.05% to 0.1%. Sodium Azide acts as a metabolic inhibitor by targeting the cell’s energy-producing pathways. This inhibition prevents the cells from internalizing or shedding the surface markers that the fluorescent antibodies are binding to, ensuring the signal remains stable until the moment of analysis. Sodium Azide also helps prevent bacterial contamination over short storage periods.
Specialized Buffer Modifications
The basic FACS buffer formula is frequently adapted to meet the demands of specific experiments. Many standard protocols use PBS that specifically excludes calcium and magnesium ions. These divalent cations can promote cell-to-cell adhesion, causing clumping that disrupts the single-file analysis required by the flow cytometer.
In cases where cells are prone to clumping, a chelating agent like Ethylenediaminetetraacetic acid (EDTA) (0.5 to 5 mM) may be added to bind remaining divalent cations. Conversely, if an experiment involves studying receptors that require calcium or magnesium for function, the buffer must be modified to include these ions, often necessitating a non-chelating salt solution like Hanks’ Balanced Salt Solution (HBSS).
A major modification involves preparing cells for intracellular staining, which requires temporary disruption of the cell membrane. For this purpose, the buffer is supplemented with detergents like Triton X-100 or saponin, creating a permeabilization buffer. This allows large antibody molecules to pass through the membrane and bind to targets within the cytoplasm or nucleus. Furthermore, when cells are sorted for later functional use, Sodium Azide is often omitted, as its metabolic inhibition would compromise the long-term viability of the collected cells.