Flow Cytometry Course

Lesson 1 • Signal Generation and Detection

Covers how photons are converted to electronic signals by detectors and amplified for analysis. Links detector type and gain settings to sensitivity and dynamic range.

Lesson 2 • Principles of Light and Optics

Explains how light interacts with particles to produce scatter and fluorescence signals. Provides the optical foundation needed to understand detector placement and filter selection.

Lesson 3 • Overview of Data Parameters

Introduces forward scatter, side scatter, and fluorescence channels as the primary measured parameters. Prepares students to interpret dot plots and histograms in later chapters.

Lesson 4 • History and Core Applications

Traces flow cytometry from early cell counters to modern multiparameter platforms. Establishes clinical, research, and industrial contexts that motivate all subsequent technical learning.

Lesson 1 • Cell Sorter Mechanics

Introduces electrostatic and microfluidic sorting mechanisms that physically separate target populations. Distinguishes sorter configurations by purity, yield, and throughput trade-offs.

Lesson 2 • Instrument Quality Control Practices

Establishes daily and periodic QC routines using calibration beads and standardized protocols. Ensures students can verify instrument performance before every experiment.

Lesson 3 • Laser Sources and Beam Shaping

Examines laser types, power levels, and beam-shaping optics used in modern cytometers. Connects laser choice to fluorochrome excitation efficiency and panel design.

Lesson 4 • Detector Arrays and Electronics

Maps the path from photon collection through detector arrays to digitized data output. Grounds students in voltage, gain, and threshold settings that control data acquisition.

Lesson 1 • Fluorochrome Photophysics

Explains absorption spectra, quantum yield, and extinction coefficient as determinants of fluorochrome brightness. Provides the quantitative basis for comparing reagents across panels.

Lesson 2 • Panel Design Workflow

Provides a systematic process for matching fluorochromes to targets, instruments, and experimental goals. Students apply brightness hierarchy and antigen density rules to build optimized panels.

Lesson 3 • Antibody Conjugate Quality

Addresses fluorochrome-to-protein ratio, lot-to-lot variability, and storage conditions that affect conjugate performance. Teaches students to evaluate and validate antibody reagents before use.

Lesson 4 • Spectral Overlap and Spillover

Quantifies how emission from one fluorochrome contaminates adjacent detector channels and why compensation is required. Prepares students for the compensation procedures covered in the next chapter.

Lesson 1 • Cell Viability Assessment

Teaches exclusion and fixable viability dye strategies to gate out dead cells that cause false positives. Connects viability gating to downstream data accuracy.

Lesson 2 • Biological Sample Types and Handling

Compares preparation requirements for whole blood, bone marrow, solid tissue, and cell culture samples. Establishes handling rules that prevent artifacts before staining begins.

Lesson 3 • Fixation, Storage, and Biosafety

Addresses post-staining fixation for sample preservation and pathogen inactivation in biosafety contexts. Ensures students comply with safe handling standards for infectious samples.

Lesson 4 • Intracellular and Nuclear Staining

Covers fixation and permeabilization chemistries required to access cytoplasmic and nuclear targets. Addresses compatibility between fixation reagents and fluorochrome stability.

Lesson 1 • Compensation Matrix Calculation

Explains the mathematical derivation of spillover coefficients and how software applies them to raw data. Enables students to detect and correct compensation errors before analysis.

Lesson 2 • Compensation Controls

Specifies the types of single-color controls required and the rules for their preparation and use. Establishes why controls must match sample staining conditions to be valid.

Lesson 3 • Voltage Optimization Strategies

Teaches systematic voltage titration using beads and cells to position populations optimally on each detector. Connects voltage settings to resolution of dim populations from background.

Lesson 4 • Acquisition Settings and Run Parameters

Covers event count targets, acquisition speed, and stop gates that ensure statistically robust data collection. Prepares students to configure acquisition templates for reproducible experiments.

Lesson 1 • Fluorescence Gating Techniques

Covers Boolean, sequential, and backgating strategies for resolving complex immunophenotyping panels. Connects gate placement to FMO controls and biological knowledge.

Lesson 2 • Data File Formats and Software

Introduces the FCS file standard and the landscape of analysis software options available to researchers. Establishes file management practices that support reproducible analysis workflows.

Lesson 3 • Scatter-Based Gating

Teaches FSC/SSC gating to isolate viable singlet cells from debris, aggregates, and dead cells. Establishes the first gate in every hierarchical analysis tree.

Lesson 4 • Statistical Outputs and Reporting

Explains percent-positive, median fluorescence intensity, and cell count metrics and their appropriate use. Teaches students to select and report statistics that match their experimental question.

Lesson 1 • Bead-Based Multiplex Assays

Introduces bead arrays for simultaneous quantification of multiple soluble analytes on a flow cytometer. Covers assay setup, standard curve generation, and data interpretation.

Lesson 2 • Apoptosis and Cell Death Assays

Integrates Annexin V, caspase, and mitochondrial membrane potential assays to distinguish apoptosis stages. Enables students to design multi-parameter death pathway panels.

Lesson 3 • Functional Assays by Flow Cytometry

Covers intracellular cytokine staining, degranulation, and phosphoflow protocols for measuring cell function. Connects stimulation conditions and fixation timing to assay sensitivity.

Lesson 4 • High-Dimensional Immunophenotyping

Extends panel design to 20-plus parameters for deep immune profiling of blood and tissue samples. Addresses the unique challenges of reagent compatibility and data complexity at high dimensionality.

Lesson 1 • Reporting and Data Presentation

Establishes standards for presenting flow cytometry data in publications, reports, and regulatory submissions. Covers figure construction, gating hierarchy display, and data transparency requirements.

Lesson 2 • Interpreting Biological Variability

Distinguishes technical noise from true biological variation in cytometric datasets. Teaches students to contextualise results within known biology and published reference ranges.

Lesson 3 • Automated and Computational Gating

Introduces clustering algorithms and automated gating tools that reduce operator bias in high-dimensional data. Evaluates when automated approaches outperform manual gating.

Lesson 4 • Statistical Analysis for Flow Data

Applies parametric and nonparametric tests appropriate for cytometric data distributions and sample sizes. Addresses multiple comparison corrections and effect size reporting.