Flow Cytometry Sample Preparation

What is Flow Cytometry?

Flow cytometry is a sophisticated laser-based technique employed to evaluate the expression of cell surface and intracellular molecules. Central to both research and diagnostics, it enables the simultaneous multi-parameter analysis of individual cells, aids in characterizing varying cell types in mixed populations, and can measure cell size and volume. Flow cytometry assays determine the purity of isolated cell subgroups and even sort diverse cell populations, a process termed fluorescence-activated cell sorting (FACS). This is achieved by detecting fluorescence intensity emanating from cells labeled with fluorescent antibodies specific to cell proteins or ligands, like propidium iodide linked to DNA.

What is a Flow Cytometer?

Depending on its configuration and the intended application, a flow cytometer can have various components and capabilities including:

• Fluidics System: This system transports cells in a single file, ensuring that only one cell at a time passes through the laser beam for analysis.
• Lasers: These are used to illuminate the cells as they pass through the beam. Depending on the configuration of the cytometer, there may be multiple lasers with different wavelengths to allow for simultaneous detection of multiple fluorescent tags.
• Optics System: This system detects scattered light and fluorescence. It includes lenses and filters to ensure that the appropriate light is detected.
• Detectors: These convert the light signals into electronic ones. Common detectors include photodiodes for forward scatter (FSC) and photomultiplier tubes (PMTs) for side scatter (SSC) and fluorescence.
• Electronics System: This interprets the signals received from the detectors and translates them into data points that can be visualized and analyzed.

Methods for Flow Cytometry Sample Preparation

The sample preparation for flow cytometry involves creating a single-cell suspension, typically from cell cultures or tissue samples, and incubating these cells with either unlabeled or fluorophore-tagged antibodies. Here are some common methods of FC Sample Preparation:

• Density Gradient Centrifugation: This method is used to isolate specific cell populations, such as lymphocytes, from heterogeneous samples like blood.
• Direct Immunofluorescence Staining: Cells are directly stained with fluorochrome-conjugated antibodies.
• Indirect Immunofluorescence Staining: Cells are first stained with an unconjugated primary antibody and then with a fluorochrome-conjugated secondary antibody that recognizes the primary antibody.

Why is Flow Cytometry Sample Preparation so difficult:

• Cell Viability: Maintaining cell health and viability during the preparation process is crucial but can be challenging.
• Specificity of Staining: Ensuring that the fluorescent labels or antibodies bind specifically without non-specific staining or background fluorescence.
• Sample Heterogeneity: Dealing with samples that have a mixture of cell types or states can complicate the preparation process.

Benefits of Automation over Manual Pipetting for Flow Cytometry Sample Preparation:

• Consistency: Automation ensures uniform sample treatment, reducing variability and improving repeatability.
• Efficiency: Automated systems can handle multiple samples simultaneously, streamlining the preparation process.
• Reduced Sample Loss: Automated devices can be more precise in handling samples, leading to minimal losses.
• Reliability: Automation reduces human errors and ensures a standardized approach to sample preparation.