Flow cytometry is a laboratory technique used to analyze individual cells or particles suspended in a fluid. It rapidly measures their physical and chemical characteristics as they pass through a laser beam. This technology is widely applied in various scientific fields, including immunology, cancer biology, and infectious disease monitoring, to characterize cell populations. Cell fixation is a preparatory step in flow cytometry, designed to preserve cells in a stable state for accurate and reproducible results.
Why Fix Cells
Cell fixation serves several important purposes in flow cytometry experiments. A primary reason is to preserve cellular morphology and internal structures, preventing cells from degrading or changing characteristics between sample collection and analysis. This is particularly useful for long-term studies or when immediate analysis is not possible.
Fixation also plays a safety role by inactivating potential biohazards within cell samples, especially important with patient-derived samples that might contain infectious agents. It also prepares cells for subsequent staining, particularly for intracellular targets like cytokines or phosphorylated proteins. By stabilizing the cell membrane and internal components, fixation allows for permeabilization, which creates openings for antibodies to access targets inside the cell.
Standard Fixation Reagents and Procedures
The most common fixatives in flow cytometry are formaldehyde-based solutions, such as paraformaldehyde (PFA), and alcohols like methanol. Formaldehyde cross-links proteins, stabilizing cellular structures and preventing degradation. This makes it suitable for preserving cell surface antigens and for subsequent intracellular staining. PFA is typically used at concentrations ranging from 0.5% to 4% in phosphate-buffered saline (PBS).
Methanol is a precipitating fixative that coagulates proteins and dissolves lipids. This effectively permeabilizes the cell membrane, allowing direct access for antibodies to intracellular targets without a separate permeabilization step. Methanol fixation is often preferred for analyzing DNA content or certain nuclear antigens and phosphoproteins.
A general fixation procedure involves washing cells, adding fixative, incubating, and then washing again to remove excess. For PFA, cells are typically incubated on ice or at room temperature for 15 to 30 minutes, followed by washing with PBS. For methanol, cells are often incubated in cold methanol at -20°C for about 10-15 minutes. After fixation, samples can usually be stored in a refrigerator for several days, or weeks for alcohol-fixed cells, before flow cytometric analysis. Ensure cells are in a single-cell suspension before adding fixative to prevent clumping.
Optimizing Fixation for Specific Applications
Optimizing fixation parameters is often necessary for high-quality results, as different cell types and target antigens may require specific conditions. Key variables include fixative concentration, incubation time, and temperature. For instance, while 1-4% PFA is common, lower concentrations (e.g., 0.5-1%) at cooler temperatures (4-8°C) for shorter durations (around 10 minutes) can sometimes better preserve cell structure and antibody binding. Over-fixation, especially with PFA, can increase cellular autofluorescence and potentially mask or alter epitopes, making antigen detection more challenging.
Permeabilization usually follows fixation, particularly when staining intracellular antigens using formaldehyde-based fixatives. Since formaldehyde cross-links proteins but does not create pores in the cell membrane, a separate permeabilization step is needed for antibodies to enter the cell. Permeabilization agents, such as detergents like Saponin, Triton X-100, or Tween 20, disrupt the cell membrane. The choice of agent depends on the target’s subcellular location; for example, milder detergents like Saponin are suitable for cytoplasmic antigens, while stronger ones like Triton X-100 might be needed for nuclear targets.
When performing both surface and intracellular staining, stain cell surface antigens first, then fix, and finally permeabilize for intracellular staining. This sequence helps maintain the integrity and accessibility of surface antigens, which can sometimes be affected by fixation and permeabilization treatments. For delicate cell types, such as primary cells, use gentler fixation conditions to minimize cell loss or damage. Compatibility between fixatives and fluorescent dyes is also important, as some fluorochromes, particularly protein-based ones like PE or APC, can be denatured by alcohol-based fixatives.