Blog
Insights on flow cytometry analysis, AI-assisted gating, and reproducibility in research.
Reading a CFSE Proliferation Assay: Division Index, Proliferation Index, and % Divided
Compute Division Index, Proliferation Index, and percent divided from CFSE generation peaks, with a full worked example and the precursor math shown.
Spectral Unmixing Gone Wrong: Common Problems and How to Diagnose Them
Five spectral unmixing problems that pass without an error: autofluorescence, mismatched reference controls, and near-identical spectra, with fixes.
Tandem Dye Degradation: Why PE-Cy7 and APC-Cy7 Drift Between Runs
Why PE-Cy7 and APC-Cy7 tandem dyes degrade, what the spillover drift looks like, and the controls and storage that keep compensation valid.
When to Trust a FlowSOM Cluster: Interpreting Unsupervised Clustering in Flow Cytometry
A decision framework for reading FlowSOM clusters: when back-gating, marker definition, and stability say a metacluster is real or an artifact.
Absolute Cell Counts in Flow Cytometry: The Counting-Bead Formula, Worked Through
How to calculate an absolute cell count in flow cytometry with counting beads: the formula, a worked CD4 example, and the pitfalls that bias it.
Log vs Biexponential Scale in Flow Cytometry: Why Negatives Smear Against the Axis
Why a log scale misrepresents compensated negatives in flow cytometry and when biexponential or logicle display is the right axis to use.
Median vs Geometric Mean Fluorescence Intensity: Which to Report and When
Median vs geometric mean fluorescence intensity in flow cytometry: how each behaves on skewed data and which to report for reproducible results.
MIFlowCyt Reporting: What a Minimum Information Flow Cytometry Submission Requires
What MIFlowCyt (minimum information about a flow cytometry experiment) requires for publication, and how to assemble it from your FCS files.
Spillover Spreading Error: Why Compensation Doesn't Fix Multicolor Panel Resolution
Spillover spreading error explained: why compensation leaves dim populations unresolved, and how to use the spreading matrix in panel design.
Reading an Annexin V / 7-AAD Apoptosis Assay: Quadrant Gating and the Numbers That Mislead
Annexin V / 7-AAD apoptosis quadrant gating in flow cytometry: what each quadrant means and which percentages mislead, with a worked readout.
Compensation Beads vs Cells: Which Single-Color Control to Use and When
Compensation beads vs cells for flow cytometry single-color controls: brightness, autofluorescence, and a verdict by sample and panel type.
Gating an Intracellular Cytokine Staining Experiment: From Stimulation to Cytokine-Positive T Cells
Gate an intracellular cytokine staining experiment step by step: viability dump, lineage gates, and setting the cytokine gate on a control.
PMT Voltage Optimization: Walking Through Voltration and the Peak-2 Method
PMT voltage optimization by voltration: a worked peak-2 rCV curve, the rSD floor, and stain-index confirmation for flow cytometry detectors.
Gating Regulatory T Cells: A CD25, CD127, and FoxP3 Strategy That Survives Review
A reproducible Treg gating strategy in flow cytometry: CD4, CD25 vs CD127, and FoxP3 confirmation, with FMO-anchored boundaries.
Back-Gating in Flow Cytometry: How to Validate Your Gating Strategy
Back-gating in flow cytometry: project a population onto upstream plots to catch gates that clip or contaminate, and validate your gating strategy fast.
Boolean Gating in Flow Cytometry: AND, OR, and NOT Combination Gates
How to build AND, OR, and NOT Boolean combination gates in flow cytometry for co-expression, plus the compensation trap that fakes double-positives.
When to Use a Dump Channel in Flow Cytometry (and What to Put in It)
When a dump channel helps in flow cytometry and what to put in it: a decision guide for rare-cell gating, channel scarcity, and spreading-error cost.
Autofluorescence in Flow Cytometry: Why It Happens and How to Reduce It
Why cells autofluoresce in flow cytometry and five common mistakes that cause false positives, with the fix for each: controls, viability, channel choice.
Spectral Flow Cytometry Panel Design: Reading the Similarity Index
How to read the similarity index in spectral flow cytometry panel design: thresholds, a worked four-fluorochrome matrix, and which pairs to reject.
Phospho-Flow Surface Marker Loss: Which Methanol Permeabilization Route
Methanol permeabilization strips specific surface markers in phospho-flow. Three routes to keep both readouts, plus the clone and dye lists that decide.
Flow Cytometry Sort Purity and Yield: Calculation and Post-Sort Check
Sort purity and yield are different calculations. Both formulas worked through with real numbers, plus how many events a post-sort check actually needs.
Cell Cycle Analysis by Flow Cytometry: Fitting the Dean-Jett-Fox Model and Reading the CV
Fit the Dean-Jett-Fox model to a DNA histogram, then read goodness-of-fit and the G0/G1 CV before trusting cell cycle phase percentages.
When to Use FMO Controls in Flow Cytometry (and When Isotype Controls Won't Do)
FMO controls vs isotype controls: when each one belongs in your gating workflow, what they actually measure, and when to skip both.
Over-Compensation and Under-Compensation in Flow Cytometry: How to Spot Them
Seven compensation mistakes that pass auto-calculation: smeared negatives, tandem dye drift, and how to spot them before publishing.
Doublet Discrimination in Flow Cytometry: FSC-A vs FSC-H and SSC-W Gates
How to draw the singlet gate using FSC-A vs FSC-H, where it sits in your gating hierarchy, and what to do when only SSC-W is available.
Daily Instrument QC for Flow Cytometry: When and How to Use Calibration Beads
CS&T, Rainbow, and EQC beads for daily flow cytometry calibration: which to use, specific thresholds, and the daily startup procedure.
How to Find the Right Antibody Concentration for Flow Cytometry: Titration Protocol
Antibody titration protocol for flow cytometry: calculate Stain Index, run a serial dilution series, and find the optimal concentration for your panel.
Flow Cytometry QC: Tracking Instrument Performance Across Runs
Track flow cytometry QC metrics across instrument runs using Levy-Jennings charts. Specific thresholds, Westgard rules, and PMT voltage trend tracking.
Live/Dead Discrimination in Flow Cytometry: DAPI, PI, Fixable Dyes Compared
Cell viability gating DAPI vs live-dead stain — PI, 7-AAD, DAPI, and fixable amine dyes compared on channel, fixation, brightness, and cost.
Sharing Flow Cytometry Analysis Across Labs: Workspace Files and Reproducibility Pitfalls
FlowJo workspace file collaboration across labs — 7 pitfalls that break gates in transit and the open-format fixes that survive the journey.
Exporting Flow Cytometry Statistics for Papers: MFI, Percent Positive, and Gate Labels
How to export flow cytometry statistics for publication — MFI definitions, gate labels, Prism, Gating-ML 2.0, and MIFlowCyt — without reviewer churn.
Spectral vs Conventional Flow Cytometry: When the Upgrade Is Worth It
Spectral vs conventional flow cytometry: panel size, autofluorescence, cost, and a per-reader-type verdict on when the upgrade is worth it.
Reducing Gating Variability Between Operators: Templates, FMOs, and Automated Approaches
Reducing gating reproducibility variance between operators: FMOs, locked templates, automated gating, and concordance audits with concrete numbers.
How to Identify Cell Populations by Scatter Plot: An FSC/SSC Reference
How to identify cell populations by scatter plot: FSC and SSC reference for PBMCs, whole blood, bone marrow, and the limits of scatter-only gating.
Batch Processing FCS Files: A Practical Workflow for Multi-Sample Analysis
Batch process FCS files across multiple samples without silent failures: parameter mapping, compensation drift, template traps, and validation checks.
t-SNE and UMAP for Flow Cytometry: Reading High-Dimensional Cell Populations Correctly
t-SNE and UMAP flow cytometry dimensionality reduction: prerequisites, parameter tuning, and the interpretation pitfalls practitioners miss.
How to Analyze Rare Cell Populations in Flow Cytometry: Event Count, Gates, and Statistical Confidence
How to analyze rare cell populations in flow cytometry: event count math, Poisson CV formulas, dump channel gating, worked HSC and CTC examples.
Multicolor Flow Cytometry Panel Design: Fluorochrome Selection, Controls, and Spillover Rules
How to design a multicolor flow cytometry panel: markers, lasers, strength-matching fluorochromes, SSM-aware refinement, and FMO vs isotype controls.
How to Analyze FCS Files Without FlowJo: Free Tools, Programmatic Options, and What You Give Up
Practical guide to analyzing FCS files without a FlowJo license. Covers the FCS file format structure, free tools (FCS Express, Floreada, FCSalyzer), programmatic options (R flowCore, Python FlowKit), and what you actually give up when switching from FlowJo.
Flow Cytometry Data Analysis Software Comparison: A Decision Framework for Research, Clinical, and GxP Labs
Practical flow cytometry data analysis software comparison for 2025. Decision tree for research, clinical, and GxP labs comparing FlowJo, FCS Express, Kaluza, OMIQ, and R/Bioconductor by regulatory compliance, workflow fit, and total cost of ownership.
How to Compensate for Spectral Overlap in Flow Cytometry: Controls, Matrix Math, and Validation
Step-by-step guide to compensating for spectral overlap in flow cytometry. Includes a worked spillover matrix example with real coefficients, single-color control setup, compensation validation checks, and troubleshooting common errors.
AI-Assisted Gating in Flow Cytometry: What Is Real and What Is Hype
When does AI-assisted gating actually help in flow cytometry? Decision framework for automated gating approaches, accuracy benchmarks from recent studies, and what you need in place before adopting ML-based gating.
How to Set Up a Gating Strategy in Flow Cytometry: Sequential Gates, Back-Gating, and Reproducibility
Step-by-step guide to building a reproducible flow cytometry gating strategy. Covers time gates, scatter gating, doublet discrimination, FMO controls, back-gating validation, and troubleshooting common gating errors.
FlowJo Alternatives: What Core Facilities Are Actually Switching To
Comparing free and paid FlowJo alternatives for flow cytometry analysis. Feature matrix, switching criteria, and what actually matters for core facilities evaluating new software.