Fluorescence-Activated Cell Sorting (FACS) is a laboratory technique used to separate and analyze cells based on their characteristics. A FACS buffer is a specialized liquid solution that provides a stable environment for cells during this process. It helps maintain their physiological state for accurate analysis and successful isolation. This buffer suspends cells, allowing them to pass through the instrument smoothly.
Role of Buffers in Cell Sorting
Buffers play a foundational role in biological systems by resisting changes in pH, a measure of acidity or alkalinity. In cell sorting, maintaining a stable pH is important because cellular processes are highly sensitive to pH fluctuations. Significant deviations can disrupt enzyme activity, protein structure, and overall cellular function. A properly formulated buffer ensures cells remain in a consistent chemical environment throughout sorting.
Beyond pH regulation, the buffer also maintains isotonic conditions. Isotonicity refers to balanced osmotic pressure, where solute concentration inside and outside the cell is similar. This balance prevents cells from swelling and bursting (lysis) or shrinking from water loss, which compromises their integrity. By controlling these factors, the buffer protects cells during sorting.
Common Components of FACS Buffer
A standard FACS buffer uses a base solution like Phosphate-Buffered Saline (PBS), often without calcium and magnesium ions. The absence of these divalent cations prevents cell clumping, ensuring a single-cell suspension. PBS provides a physiological salt concentration, mimicking the natural cellular environment and maintaining osmotic balance.
Proteins like Bovine Serum Albumin (BSA) (0.1-1%) or Fetal Bovine Serum (FBS) (1-10%) are added to the buffer. These proteins prevent non-specific binding of antibodies to cells or cells to surfaces. They also reduce cell aggregation, improving recovery and sorting efficiency.
Chelating agents, most commonly Ethylenediaminetetraacetic acid (EDTA) at concentrations around 0.5-5 mM, are included to prevent cell clumping. EDTA works by binding to divalent cations, which are involved in cell adhesion mechanisms. Antimicrobial agents like sodium azide (0.05-0.1%) may be added to inhibit bacterial growth during storage, though their use depends on downstream applications. For samples with a high presence of dead cells, DNase I (25-50 µg/mL) can be added to break down extracellular DNA that causes aggregation.
Maintaining Cell Integrity and Viability
FACS buffer composition impacts cell integrity and viability during sorting. Without stable pH, cells experience stress, leading to changes in surface proteins or internal structures. This can compromise sorting accuracy based on specific markers.
Proper osmolarity is important; too dilute a buffer causes cells to swell and burst, while too concentrated a buffer causes them to shrink. Both scenarios result in non-viable cells unsuitable for further analysis or culture. Proteins and chelating agents contribute to maintaining a single-cell suspension, which is essential for precise sorting and preserving isolated cell health for downstream applications.
Preparation and Handling Considerations
Preparing FACS buffer involves using high-quality, sterile reagents and deionized water to prevent contamination. After combining components, the pH must be accurately adjusted to the physiological range (typically 7.0-7.4). This adjustment is important for maintaining cell health.
Sterile filtration, often using a 0.22 µm filter, is performed to remove particulate matter and microbial contaminants, ensuring the buffer is clean for sensitive cell handling. Once prepared, the buffer should be stored under appropriate conditions, such as refrigeration at 4°C, to maintain its stability and prevent degradation of components. Proper handling and storage are important for the buffer’s effectiveness and the reliability of cell sorting experiments.