CALCULATE WITH CONFIDENCE

Flow Cytometry & Cell Sorting

Flow Cytometry Stain Index Workbench

Calculate one stain index from an explicit negative-population spread, use a separate P5-to-P95 percentile-width convention, or compare multiple entered records under one declared convention.

Biology · experimental measurements

Keep fluorescence centers, negative-population spread, denominator convention and comparative records visible without inferring gates, staining quality or an optimal titer.

Private calculations in your browser · explicit inputs and model boundaries
Example preview · 2× robust SDEntered separation relative to the scaled negative-population width
005000251e4501.5e4752e4100Entered stain-index endpoint: 100, 20,000Multiplier × negative spreadPositive center − negative center

The endpoint uses the actual entered denominator width and center separation. Its signed slope is 200; the diagram does not represent an event distribution or gate.

  1. 1EnterProvide the known values
  2. 2CalculateResults update automatically
  3. 3VerifyReview the details and units
Try an example

Enter the spread already calculated for the declared negative population; keep its exact definition with the record.

Calculation result

—

Enter valid values to see the result.

Your entries are calculated in this browser and are not submitted to 365CALCS.COM.

Feedback

Understand the relationship

The reasoning behind the result

The numerator is an entered center separation

D = centerpositive − centernegative

Subtracting the negative-population center removes the entered background center from the positive-population center. The difference remains signed.

A median and a mean summarize a population differently. The interface therefore records which center statistic the visitor used instead of labeling every value generically as MFI.

The denominator definition is part of the result

SI = D/(m × spreadnegative)

A widely used convention divides by two times the negative population's robust standard deviation. Other publications or laboratory records may use ordinary SD, one spread unit or another declared width.

Changing the spread statistic or multiplier changes the numerical index even when the fluorescence centers are unchanged, so both are exposed in the ledger.

The percentile-width form is a separate convention

SIpercentile = 3.29(P50positive−P50negative)/(P95negative−P5negative)

This form uses the entered central 90% negative-population width rather than an entered SD or robust SD. Its 3.29 factor belongs to that selected convention.

The workbench does not convert one convention into another or claim that differently defined indices are directly interchangeable.

Comparable arithmetic still needs comparable measurements

Detector scaling, fluorescence transformation, compensation, gating, negative-control material, event counts and acquisition settings can change the entered centers and spreads.

A numerical comparison does not prove that conditions from different runs, panels or instruments have equivalent measurement context.

Stain index and spillover spreading answer different questions

Stain index relates a positive-to-negative center separation to the spread of a declared negative population in the measured parameter.

A spillover-spreading matrix describes spread introduced in secondary parameters by spectral overlap and measurement statistics. This workbench does not calculate that matrix or combine the two quantities into a panel score.

Follow the numbers

Compare three entered titration records under one convention

  1. Select median centers, robust SD and a denominator multiplier of two for every record.
  2. For 0.25 µg/test, calculate (8,100−100)/(2×80) = 50.
  3. For 0.5 µg/test, calculate (12,100−100)/(2×75) = 80.
  4. For 1.0 µg/test, calculate (15,100−100)/(2×100) = 75.
  5. Retain the three numerators, robust SD values, denominators and signed indices in the comparison ledger.

The entered 0.5 µg/test record has the largest numerical index in this example, but condition selection still depends on the plots, controls, required resolution, background, reagent use and experimental constraints outside the calculation.

Quick guide

How to use this calculator

  1. Choose the formula convention that matches the source record; do not substitute ordinary SD, robust SD, another spread measure or percentile width without changing the declared selection.
  2. Enter fluorescence summaries obtained from externally established positive and negative gates under one declared transformation and detector context.
  3. For comparisons, apply one center statistic, spread statistic and multiplier across every row and retain the displayed components with each condition.
  4. Use the output as a transparent descriptive record alongside plots, gates, controls, acquisition settings and the experimental question rather than as an automatic decision rule.

Calculation method

Calculation and interpretation

Keep fluorescence centers, negative-population spread, denominator convention and comparative records visible without inferring gates, staining quality or an optimal titer.

Entered-spread convention: SI = (positive center − negative center)/(m × negative spread), with explicit multiplier m. Percentile-width convention: SI = 3.29 × (positive P50 − negative P50)/(negative P95 − negative P5).

Worked example

Compare three entered titration records under one convention

The entered 0.5 µg/test record has the largest numerical index in this example, but condition selection still depends on the plots, controls, required resolution, background, reagent use and experimental constraints outside the calculation.

Entered-spread convention: SI = (positive center − negative center)/(m × negative spread), with explicit multiplier m. Percentile-width convention: SI = 3.29 × (positive P50 − negative P50)/(negative P95 − negative P5).

Supported inputs

Precision and limits

Entered summaries only

The workbench does not read FCS files, transform events, draw gates, calculate percentiles from events or decide which populations are positive and negative.

No universal formula definition

Robust SD, ordinary SD, other spread measures, denominator multipliers and percentile-width conventions remain explicitly separate; the page does not claim they are interchangeable.

No titer or panel recommendation

The largest entered numerical index is not automatically the optimal antibody concentration, fluorochrome, detector setting or panel assignment.

No assay-quality threshold

No index value is labeled acceptable, sensitive, resolved, failed or validated, and the calculator supplies no universal cutoff.

No automatic cross-run comparability

Differences in gating, controls, transformation, compensation, detector settings, reagent lots, samples or instruments remain external limitations.

No uncertainty or population inference

The output is point arithmetic from entered summaries and includes no event-level uncertainty, replicate model, confidence interval or biological-population conclusion.

No clinical interpretation

The index does not identify cells, diagnose a specimen, establish antigen expression or support a clinical decision.

Continue calculating

Related calculators