Proliferation Index Calculator

Calculate proliferation index, CFSE dilution generations, and Ki67 staining interpretation.

Generation 0 peak MFI (CFSE undivided) (MFI)

Generation 1 MFI (MFI)

Cells in generation 0 (cells)

Cells in generation 1 (cells)

Cells in generation 2 (cells)

Cells in generation 3+ (cells)

Please check your inputs and try again.

How CFSE Dilution Works

CFSE binds cytoplasmic proteins covalently and is distributed roughly equally between daughter cells at each division. Fluorescence therefore halves with every division, producing discrete peaks.

Generation	Relative MFI	Cells from one original
0	100%	1
1	50%	2
2	25%	4
3	12.5%	8
7–8	<1%	Detection limit

Resolution runs out after seven or eight divisions, when signal approaches autofluorescence. Beyond that, peaks merge and counting is unreliable.

Two Different Indices

Metric	Denominator	Answers
Division index	All cells	Average divisions per original cell
Proliferation index	Responding cells only	Average divisions among those that divided
% divided	—	What fraction responded at all

The distinction matters when interpreting suppression. If a drug reduces the fraction responding but the responders divide normally, the division index falls while the proliferation index does not — indicating fewer cells were recruited rather than slower division. Reporting only one index conflates the two mechanisms.

Why Cell Numbers Must Be Back-Calculated

Each generation contains 2ⁿ cells per original precursor, so observed counts must be divided by 2ⁿ to recover how many cells started in each generation. Skipping this step massively overweights the later generations, since one original cell that divided three times contributes eight events.

Confound	Effect
Cell death	Later generations lost, understating division
Uneven dye partitioning	Peak broadening, merged generations
Dye toxicity at high concentration	Suppresses the proliferation being measured
Autofluorescence	Sets the practical resolution limit

CFSE at excessive concentration is itself antiproliferative, so titrating to the lowest workable level is standard. Compared with Ki-67, which marks any cell in cycle at one moment, CFSE gives division history — the two answer different questions.

Worked Examples

Example 1: T cell activation: G0=100, G1=200, G2=300, G3=150

precursors=100,100,75,18.8 → % divided=66.0%

Result: Division index=1.04, Proliferation index=1.58

Robust T cell proliferation to mitogen

Example 2: Suppressed: G0=400, G1=100, G2=50, G3=10

precursors=400,50,12.5,1.25 → % divided=13.7%

Result: Regulatory T cell or drug suppression

Treg-mediated suppression of effector cells

Example 3: Suppression of recruitment versus rate

Division index falls, proliferation index unchanged

Result: Fewer cells recruited

The cells that did respond divided normally. The drug reduced how many entered division rather than how fast they divided.

Example 4: Why back-calculation matters

400 cells in generation 3

Result: 50 original precursors

Each precursor produced eight cells. Counting raw events would weight this generation eight times too heavily.

Common Mistakes

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Not dividing counts by 2ⁿ

Each original cell dividing n times produces 2ⁿ events. Without back-calculation, later generations dominate the result.

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Reporting only one index

Division index and proliferation index distinguish fewer responders from slower division. Reporting one alone conflates the mechanisms.

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Using CFSE beyond seven or eight divisions

Signal approaches autofluorescence and peaks merge. Counting becomes unreliable past that point.

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Overloading with dye

High CFSE concentrations are themselves antiproliferative, suppressing the response being measured. Titrate to the lowest workable level.

Frequently Asked Questions

CFSE dilution principle?

CFSE (carboxyfluorescein diacetate succinimidyl ester) covalently labels cell proteins. With each division, fluorescence halves (dye partitioned equally between daughters). Flow cytometry resolves peaks. Each peak = one generation. Label index: fraction of original dye remaining.

Ki67 vs CFSE?

Ki67: nuclear protein expressed in all active phases (G1, S, G2, M), absent in G0. Simpler: one antibody, no pre-labeling. But: only snapshot (are cells cycling NOW?). CFSE: tracks history of division. Both used routinely for proliferation assessment.

How does CFSE track division?

The dye binds cytoplasmic proteins and partitions equally between daughter cells, so fluorescence halves at each division, producing discrete peaks.

What is the difference between division and proliferation index?

Division index averages over all cells including non-responders; proliferation index averages only over cells that divided at least once.

Why divide counts by 2ⁿ?

Because one original cell dividing n times yields 2ⁿ cells. Back-calculation recovers the number of precursors in each generation.

How many divisions can CFSE resolve?

Seven or eight, after which fluorescence approaches autofluorescence and generation peaks can no longer be separated.