Flow cytometry is a powerful analytical technique used in biomedical research, clinical diagnostics, and various other fields to analyze and quantify characteristics of individual cells in a heterogeneous population. This method provides detailed information about the physical and chemical properties of cells, allowing researchers and clinicians to gain insights into cell structure, function, and behavior. The flow cytometry process involves suspending cells in a fluid and passing them through a narrow, focused stream of this fluid. As each cell passes through a laser beam, it scatters light in different directions and emits fluorescence if labeled with fluorochrome-conjugated antibodies or other fluorescent markers. The scattered and emitted light is then collected by detectors, generating data that can be used to characterize and differentiate cells based on various parameters. Key features of flow cytometry include: Cellular Analysis: Flow cytometry enables the analysis of multiple parameters for each individual cell, such as size, granularity, and fluorescence intensity. This allows for the identification of different cell types within a complex mixture. Fluorescent Labeling: Cells can be labeled with fluorochrome-conjugated antibodies or other fluorescent dyes that specifically bind to cellular components, such as surface markers, intracellular proteins, or nucleic acids. This allows for the identification of specific cell populations or the quantification of particular molecules within cells. High Throughput: Flow cytometry is capable of analyzing thousands of cells per second, providing rapid and high-throughput data acquisition. Cell Sorting: In addition to analysis, flow cytometry can be coupled with cell sorting technologies, allowing the isolation of specific cell populations based on defined criteria. This is particularly useful for downstream experiments or therapeutic applications. Multiparametric Analysis: Flow cytometry allows the simultaneous measurement of multiple parameters for each cell, providing a multidimensional view of cellular characteristics. Applications of flow cytometry are diverse and include immunophenotyping of cell populations, cell cycle analysis, apoptosis detection, DNA content analysis, and functional assays. In clinical settings, flow cytometry is often used for diagnosing and monitoring various diseases, including hematological malignancies and immunodeficiency disorders. Overall, flow cytometry plays a crucial role in advancing our understanding of cellular biology and has become an indispensable tool in many areas of research and diagnostics.

1. Sysmex Cube 6 | Basics of Flowcytometry
Presented by: Amit Kr Bhardwaj | Chimera Labs

2. What is Flowcytometry ?
 Flowcytometry helps in measuring physical and chemical properties
of cells or cell like particles in suspension when they pass one by
one in front of a laser beam at a rapid pace.
The power of flow cytometry comes from its ability to analyze
multiple physical and chemical characteristics simultaneously on a
large numbers of events in a heterogeneous sample. Using these
measurements specific subpopulations can be identified.
It provides information on cell size, complexity, and the expression
of specific molecules on the cell surface.

3. Components of Flowcytometry

4. Hydrodynamic Focusing
• Alignment: Hydrodynamic focusing aligns
particles or cells within a narrow central channel
using a sheath fluid, ensuring precise positioning
during flow cytometry analysis.
• Benefits: It enhances measurement precision,
sensitivity, and data quality by reducing
interference between particles and allowing for
efficient data collection.

5. Forward Scatter (FSC)
• FSC Measures Size: Forward scatter quantifies the
size of particles or cells by analyzing the intensity
of light scattered in the forward direction.
• Size Discrimination: It's used to differentiate
between particles based on size, enabling the
identification of specific populations.

6. Side Scatter (SSC)
• Side scattering is caused by the granularity
and complexity inside the cell
• Granularity Indicator: SSC measures
internal granularity or complexity of
particles or cells.
• Cell Differentiation: It helps differentiate
cell types based on their internal
properties.

8. Fluorescence
• Fluorescence Principle: Fluorescence in flow cytometry
involves the detection of emitted light (fluorescence) from
fluorochrome-labeled molecules when they are exposed to
a specific wavelength of light (excitation) from a laser.
• Fluorochromes and Probes: Fluorescent dyes or antibodies
labeled with fluorochromes bind to specific targets within
cells or particles. Different fluorochromes emit light at
distinct wavelengths, enabling the identification of
multiple markers simultaneously.
• Detection Channels: Flow cytometers have multiple
detection channels, each tuned to capture fluorescence at
specific wavelengths. This allows for the simultaneous
measurement of various markers on the same cell or
particle.

9. Fluorescence Detection
I. Labeling with Fluorescent Probes: Cells or
particles are tagged with fluorescent dyes or
antibodies.
II. Laser Excitation: A laser beam excites the
fluorescent markers, causing them to absorb
energy.
III. Emission of Fluorescent Light: The excited
markers emit fluorescent light with a longer
wavelength.
IV. Detection and Analysis: Detectors capture the
emitted light, and specialized software analyzes the
data, providing insights into marker presence and
quantity.

11. Sysmex Cube 6
• Compact and economic flow cytometer
• Configurations with up 6 optical parameters (up to 4 colors)
• 488 nm blue laser and 638 nm red laser
• Particle size: 0.1 – 100 µm
• True VolumetricAbsolute Counting (TVAC)
• Aspiration speed adjustable from 0.1µLto 20µL/s
• maximum acquisition rate at 15,000 particles /second