Flow Cytometry Sample Preparation

What is flow cytometry?

Flow cytometry is a sophisticated laser-based technique used to assess the expression of molecules on and within cells. Central to research and diagnostics, it enables simultaneous parametric analysis of single cells, helps characterize different cell types in mixed populations, and measures cell size and volume. Flow cytometry assays can determine the purity of isolated cell subpopulations and even classify different cell populations, a process called fluorescence-activated cell sorting (FACS). This is accomplished by detecting the intensity of fluorescence emitted by cells labeled with fluorescent antibodies directed against cellular proteins or ligands such as propidium iodide linked to DNA.

What is flow cytometry?

Depending on its configuration and intended application, a flow cytometer can have a variety of components and features, including:

• Fluid system:  The system transports cells in a single column manner, ensuring that only one cell is analyzed through a laser beam at a time.
• Laser:  Used to illuminate cells when they pass through a beam of light. According to the configuration of the cell analyzer, there may be multiple lasers of different wavelengths to simultaneously detect multiple fluorescent labels.
• Optical system:  This system detects scattered light and fluorescence. It includes a lens and a filter to ensure the detection of appropriate light.
• Detector:  Common detectors for converting optical signals into electronic signals include photodiodes using dry forward scattering (FSC) and photomultiplier tubes (PMT) using dry side scattering (SSC) and fluorescence.
• Electronic system:  Explain the signals received from the detector and convert them into data points that can be visualized and analyzed.

Flow Cytometry Sample Preparation Methods

Sample preparation for flow cytometry involves creating a suspension of single cells (usually from a cell culture or tissue sample) and then incubating these cells with unlabeled or fluorophore-tagged antibodies. Here are some common FC sample preparation methods:

• Density gradient centrifugation:  This method is used to isolate specific cell populations, such as lymphocytes, from heterogeneous samples such as blood.
• Direct immunofluorescence staining:  Stain cells directly with antibodies bound to fluorescent dyes.
• Indirect immunofluorescence staining:  Firstly, stain the cells with unbound primary antibody, and then stain the cells with fluorescein bound secondary antibody that recognizes the primary antibody.

Why flow cytometry sample preparation is so difficult:

• Cell Viability: Maintaining cell health and viability during preparation is critical but also challenging.
• Staining specificity: Ensure specific binding of fluorescent markers or antibodies and no non-specific staining or (background) fluorescence.
• Sample heterogeneity: Handling samples with a mixture of multiple cell types or states can complicate the preparation process.

The advantages of automating flow cytometry sample preparation compared to manual pipetting are as follows:

• Consistency: Automation ensures uniform sample processing, reducing variability and improving reproducibility.
• Efficiency: Automated systems can process multiple samples simultaneously, simplifying the preparation process.
• Reduced sample loss: Automated equipment can handle samples more precisely, thus minimizing losses.
• Reliability: Automation reduces human error and ensures a standardized approach to sample preparation.