Flow Cytometry Calculator
Calculate cell count, viability, and population percentages from flow cytometry data.
Reading the Annexin V / PI Quadrants
Phosphatidylserine flips to the outer membrane leaflet early in apoptosis, where annexin V binds it. Propidium iodide enters only once membrane integrity fails. Together they resolve four populations.
| Quadrant | Annexin V | PI | Population |
|---|---|---|---|
| Lower left | − | − | Live |
| Lower right | + | − | Early apoptotic |
| Upper right | + | + | Late apoptotic or secondary necrotic |
| Upper left | − | + | Necrotic, or debris and artefact |
The upper left quadrant deserves scrutiny. Cells that are PI-positive without annexin binding are unusual biologically, so a large population there usually indicates mechanical damage during handling or gating problems rather than genuine necrosis.
Why Gating Order Matters
Analysis proceeds through a fixed sequence, and skipping a step corrupts everything downstream.
| Step | Gate | Removes |
|---|---|---|
| 1 | FSC-A vs SSC-A | Debris and dead cells outside the main population |
| 2 | FSC-A vs FSC-H | Doublets — two cells read as one |
| 3 | Viability dye | Dead cells that bind antibody non-specifically |
| 4 | Marker of interest | — |
Doublet exclusion is the step most often skipped and the most consequential. Two cells passing together produce a single event with roughly double the area but similar peak height, so plotting area against height separates them. Without this gate, a doublet of one positive and one negative cell reads as a single double-positive event — which can manufacture an entire spurious population.
Absolute Counts and Compensation
Flow cytometry gives percentages by default. Absolute counts require either counting beads at known concentration or a volumetric instrument. A rising percentage with a falling absolute count is a common and misleading pattern when one population is being depleted.
Compensation corrects for spectral overlap between fluorophores and must be set with single-stained controls on the same cell type. Over-compensation creates artefactual negative populations; under-compensation manufactures false positives.
Worked Examples
Common Mistakes
Two cells read as one event. A doublet of a positive and a negative cell appears as a genuine double-positive, creating populations that do not exist.
A percentage can rise while the absolute number falls, if another population is being depleted faster. Counting beads or a volumetric instrument gives real numbers.
Autofluorescence and antibody binding differ between cell types. Single-stain controls must match the experimental sample.
That combination is biologically unusual. A substantial upper-left population usually means handling damage or a gating error.