FACS buffer, also known as flow cytometry staining or wash buffer, is a specialized solution used to prepare, wash, and stain cells before analysis on a flow cytometer. The buffer maintains the structural integrity of the cells outside of their normal culture environment. Proper formulation directly impacts the accuracy of the final flow cytometry data. A carefully prepared buffer minimizes background noise, prevents cell clumping, and ensures cells flow smoothly through the instrument.
Essential Components and Purpose
The foundation of most FACS buffer formulations is a balanced salt solution, typically phosphate-buffered saline (PBS) or Hanks’ Balanced Salt Solution (HBSS), which maintains the physiological pH and osmotic pressure of the cells. Using PBS free of calcium and magnesium ions is standard practice because these divalent cations can promote cell-to-cell adhesion. This prevents unwanted cell aggregates that could clog the flow cytometer’s fluidics.
A protein source is included as a blocking agent. Most protocols use bovine serum albumin (BSA) at \(0.1\) to \(1.0\%\) or fetal bovine serum (FBS) between \(1\) and \(10\%\). This protein saturates non-specific binding sites on the cell surface, preventing antibodies from sticking where they are not intended to target. Reducing non-specific binding minimizes background fluorescence and increases the signal-to-noise ratio in the final data.
A chelating agent, such as ethylenediaminetetraacetic acid (EDTA), is another common additive, used from \(0.5\) to \(5.0\) millimolar (mM). EDTA binds to divalent cations, reinforcing the anti-clumping action started by using \(\text{Ca}^{2+}/\text{Mg}^{2+}\)-free PBS. This chelation ensures the cell suspension remains a single-cell preparation, which is required for accurate flow cytometry.
Standard staining buffers incorporate a preservative, most often sodium azide (\(\text{NaN}_3\)), at \(0.05\) to \(0.1\%\). Sodium azide functions as a metabolic inhibitor, temporarily halting cellular processes that could cause the internalization or shedding of cell surface proteins, thereby stabilizing the cell’s phenotype. It also inhibits bacterial growth, benefiting buffers prepared in bulk and stored long-term.
Detailed Preparation Protocol
Preparing a standard \(1\) liter batch of FACS buffer begins by calculating the volumes of concentrated stock solutions needed for the final \(1\text{X}\) working concentration. The starting point is \(1\text{X}\) \(\text{Ca}^{2+}/\text{Mg}^{2+}\)-free PBS, prepared from a \(10\text{X}\) stock solution to provide the correct salt and pH balance. For a \(1\) liter volume, \(100\) milliliters of \(10\text{X}\) PBS stock are combined with \(800\) milliliters of high-quality, deionized water.
Next, the blocking protein is added, such as \(10\) milliliters of \(20\%\) BSA stock solution to achieve a \(1\%\) final concentration in a \(1\) liter total volume. This is followed by the chelating agent; for example, \(2\) milliliters of a \(0.5\) molar EDTA stock solution would be used to reach a final concentration of \(1\) mM. The components should be mixed gently using a magnetic stirrer at a low speed to ensure full dissolution without causing foaming or protein denaturation.
The preservative, sodium azide, is then introduced, often as a \(10\%\) weight/volume stock solution, with \(1\) milliliter added to the \(1\) liter volume for a final \(0.1\%\) concentration. Because sodium azide is toxic, it must be handled in a well-ventilated area with appropriate personal protective equipment. After all components are added, the solution is brought up to the final \(1\) liter volume with deionized water, and the pH is verified to be within the physiological range of \(7.2\) to \(7.4\).
Quality Control, Storage, and Common Variations
After the FACS buffer is fully prepared, a sterile filtration step is necessary to remove particulate matter and microbial contaminants. This involves passing the buffer through a \(0.22\) micrometer pore size filter, which traps bacteria and fine debris that could clog the flow cytometer’s narrow fluidic lines. Filtering the buffer ensures instrument longevity and maintains the sterility required for experiments involving live cells.
Once filtered, the buffer is typically dispensed into manageable volumes and stored at \(4^\circ\text{C}\). At this temperature, and with the presence of sodium azide as a preservative, a well-prepared FACS buffer can maintain its integrity for up to three months. The cold temperature also helps to minimize protein degradation.
While the standard recipe is widely applicable, specific experimental needs necessitate common variations in the buffer composition. For example, if cells are intended for downstream applications like cell sorting or functional assays, sodium azide must be omitted because it is a metabolic toxin that compromises cell viability. This azide-free version is often prepared fresh or has a significantly shorter shelf life.
When dealing with cell types that have a high tendency to aggregate, the concentration of EDTA may be increased up to the \(5\) mM maximum for a stronger chelating effect. Conversely, for robust cell lines that do not easily clump, the EDTA component can be reduced or omitted entirely. For samples with a large population of dead cells that release sticky DNA, the buffer can be supplemented with DNase I and \(\text{MgCl}_2\) to digest the extracellular DNA and prevent clumping.