Flow Cytometry Calculator — Free Lab Calculators

What this is for: Four sheets — staining-cocktail builder, absolute counts from counting beads, acquisition volume/time planning, and antibody titration by stain index.

The formula: stain index = (MFI positive − MFI negative) ÷ (2 × SD of negative); cells/µL = (cell events ÷ bead events) × bead concentration

How to use:

In Staining Cocktail, enter the number of samples, overage, staining volume per test and cells per test, then list the panel as one row per reagent with its µL per test.
Read the tests to prepare, cell concentration needed, total cells and the scaled reagent and buffer volumes.
In Absolute Counts, enter cell events, bead events, bead stock concentration, bead volume and sample volume, plus the subset percentage if needed.
In Acquisition, enter sample concentration, flow rate and target events, and for a dilution enter current and target concentrations and the final volume wanted.
In Stain Index, enter one titration row per amount — µL per test, MFI positive, MFI negative and rSD negative — and use the amount with the highest SI.

Staining Cocktail & Cells per Test

Enter your panel (µL of each reagent per test), the number of samples and an overage for pipetting loss; the cocktail scales and the cell concentration needed is worked out.

Number of samples

Overage (%)%

Staining volume per test (µL)µL

Cells per testcells

Panel — one row per reagent: name, µL per test, note

Tests to prepare (samples + overage)

Cell concentration needed (cells/mL)

Total cells needed

Reagents per test (µL)

Buffer per test (µL)

Total cocktail to prepare (µL)

Total buffer (µL)

Total reagents (µL)

Absolute Counts with Counting Beads

Absolute count (cells/µL) = (cell events / bead events) × (beads added to the tube / sample volume).

Cell events acquired

Bead events acquired

Bead stock concentration (beads/µL)beads/µL

Bead volume added (µL)µL

Sample volume in tube (µL)µL

Subset (% of the counted population)%

Beads per tube

ABSOLUTE COUNT (cells/µL)

Total cells in tube

Subset absolute count (cells/µL)

Acquisition Time & Dilution to Target

Event rate = sample concentration × flow rate. Section B dilutes a stock suspension to a target concentration (C1V1 = C2V2 in cells/µL).

Sample concentration (cells/µL)cells/µL

Flow rate (µL/min)µL/min

Target events

Current concentration (cells/µL)cells/µL

Target concentration (cells/µL)cells/µL

Final volume wanted (µL)µL

Event rate (events/sec)

Time to acquire target (sec)

Time to acquire target (min)

Dilution factor

Stock suspension to take (µL)

Buffer to add (µL)

Antibody Titration & Stain Index

Stain index SI = (MFI positive − MFI negative) / (2 × rSD negative). The recommended amount is the one with the highest SI (INDEX/MATCH on the maximum, as in the workbook).

Titration — one row per amount: µL per test, MFI positive, MFI negative, rSD of negative

Highest stain index

Amount at highest SI (µL/test)

Prefer to work offline or keep a copy? The download link is right below.
Download the Flow Cytometry Calculator workbook (.xlsx)

Notes & limits (from the workbook): Antibody amounts should come from your own titration (see the Stain index section) — manufacturer 'µL per test' values are a starting point only. Keep cells/test and staining volume the same as in your titration, or the optimal amount changes. For absolute counts, acquire enough bead events (500+ minimum, 1,000+ is better); if you diluted the sample before adding beads, multiply the result by that dilution factor yourself — it is not built in. Event rates above ~10,000/sec raise coincidence/abort losses on most analysers. For the stain index, use medians, not means, and rSD (robust SD) from your analysis software.