Flow cytometry is a laboratory technique that rapidly analyzes individual cells or particles suspended in a fluid. It gathers detailed information about cell populations, including their physical characteristics and the presence of specific molecules on or within them. By measuring thousands of cells per second, flow cytometry provides a comprehensive overview of cellular properties, valuable in fields ranging from basic research to clinical diagnostics. This process allows for the identification and quantification of different cell types within a mixed sample, offering insights into cell size, internal complexity, and molecular expression.
Understanding the Core Principles
A flow cytometer operates through three main systems: fluidics, optics, and electronics. The fluidics system precisely guides cells past a laser beam in single file for individual measurement. This is achieved using a sheath fluid that surrounds the sample, creating a narrow core stream where cells flow in an orderly manner, a process known as hydrodynamic focusing. This precise alignment allows for consistent and accurate analysis of each cell.
The optical system involves lasers that illuminate cells as they pass through the interrogation point. When a cell intercepts the laser, light is scattered, and any fluorescent markers on or in the cell are excited, causing them to emit light. Detectors then capture these scattered and emitted light signals. Forward scatter (FSC) measurements indicate relative cell size, while side scatter (SSC) provides information about internal complexity or granularity.
The electronics system converts collected light signals into digital data. Photomultiplier tubes (PMTs) are commonly used detectors that transform photons into electronic pulses, where the magnitude of the current is proportional to the light intensity. This electronic data is then processed and stored by a computer, allowing for quantitative analysis of each cell’s properties. This integrated system enables the rapid collection of multiparameter data from cell populations.
Preparing Your Samples for Analysis
Before analysis, biological samples must be carefully prepared for accurate flow cytometry results. A crucial initial step involves creating a single-cell suspension, meaning cells must be separated from each other to prevent clumping, which could lead to inaccurate measurements. This might involve mechanical dissociation or enzymatic digestion, depending on the sample source like solid tissues or adherent cell lines. For suspended cells like those from blood, red blood cells may need to be lysed or peripheral blood mononuclear cells (PBMCs) isolated.
Maintaining cell viability is important, as dead or dying cells can non-specifically bind antibodies and introduce artifacts. Researchers often use viability dyes to exclude compromised cells from analysis, ensuring that only healthy cells are measured. Resuspending cells at an appropriate concentration, typically between 10^5 to 10^7 cells/mL, helps optimize instrument performance and prevents issues like missed events or prolonged run times.
Fluorescent staining is a key part of sample preparation, where specific cell markers are targeted using antibodies conjugated to fluorochromes. These fluorescent labels enable the identification and characterization of different cell populations based on their unique expression patterns. Proper controls throughout the staining process are essential for reliable data interpretation. Unstained controls provide a baseline for cellular autofluorescence and help in setting scatter parameters. Single-color controls assess spectral overlap between different fluorochromes, which is necessary for proper compensation.
Operating the Flow Cytometer
Operating a flow cytometer involves several practical steps, beginning with daily setup and quality control (QC) checks. Regular cleaning of the fluidics system is important to prevent build-up of debris that can affect performance. Calibration beads are run to verify instrument alignment and ensure consistent performance over time. These beads help in setting and monitoring detector voltages.
Once the instrument is calibrated, samples can be loaded for data acquisition. Samples are introduced into the cytometer. The flow rate can be adjusted (e.g., high, medium, or low) depending on the desired acquisition speed and resolution. Slower flow rates generally allow for better resolution.
Data acquisition parameters are set using the instrument’s software interface. This includes adjusting voltages to optimize signal detection and ensure fluorescent signals are on scale. Compensation settings are also crucial, correcting for spectral overlap. Modern flow cytometry software provides tools for real-time visualization of data as it is acquired. The software collects raw data, saving it in a standardized format.
Interpreting Your Flow Cytometry Data
After data acquisition, interpreting flow cytometry data involves specialized software to visualize and analyze the collected information. Data is commonly displayed using various plot types. Histograms are useful for single-parameter data, showing the intensity of a particular marker on the x-axis and the cell count on the y-axis.
Scatter plots are widely used for multiparameter analysis, displaying two parameters simultaneously on the x and y axes. Clusters of dots indicate distinct cell populations. Forward scatter (FSC) versus side scatter (SSC) helps in identifying different cell types based on their size and internal complexity. Density plots and contour plots are also used to visualize data density by showing regions where cells are more concentrated.
“Gating” is a fundamental process in data analysis, where regions are drawn on these plots to isolate and define specific cell populations. This allows researchers to focus on particular cell subsets for further analysis. Proper placement of these gates is important for accurate quantification of cell populations.
Compensation is a mathematical correction applied to the data to account for spectral overlap between fluorochromes. This process ensures that the signal detected in each channel truly reflects the fluorescence from its intended fluorochrome. Fluorescence Minus One (FMO) controls are important for setting accurate gates, particularly when dealing with dimly expressed markers or when fluorescence spread is significant, by showing the contribution of all other fluorochromes in the absence of one specific marker.