Photo of microspheres
  • Confidence—ensure the reliability of optimal daily instrument performance
  • Reliability—minimal variation for consistent data acquisition
  • Compatibility—full range of tools for any instrument

Instrument calibration is critical to collecting and analyzing accurate experimental data. Our flow cytometer alignment and cell sorting beads are designed to help ensure your flow cytometer is performing at its peak, your experimental design is robust, and the data you collect and analyze are accurate. In addition, our size calibration and size reference kits serve as reliable size references for flow cytometer users.

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Flow cytometer calibration beads selection guide

  Rainbow Calibration Particles AlignFlow Flow Cytometry Alignment Beads
Use Routine calibration of flow cytometers
Contents Single mixture of particles featuring 8 different fluorescent intensities. Single fluorescence, laser-specific particles available in three versions (per laser excitation).
Laser type UV to red laser UV laser Blue laser Red laser
Excitation 365–650 nm 350–370 nm 488 nm 633 nm
Emission 400–680 nm 400–470 nm 515–660 nm 645–680 nm
Bead diameter 3.0–3.4 micron 2.5 micron 6.0 micron 2.5 micron 6.0 micron 2.5 micron 6.0 micron
Cat. No. A34305 A16502 A16505 A16500 A16503 A16501 A16504
  Flow Cytometry Size Calibration Kit
Use Estimate the size of cells in an experimental sample by comparing the FSC signals with those of the reference microspheres.
Contents Contains six suspensions of unstained polystyrene microspheres (1.0 micron to 15 micron diameter).
Laser type Any
Cat. No. F13838
  Flow Cytometry Sub-micron Particle Size Reference Kit
Use Check any of the following:
  • Resolution limit and dynamic range of particle size measurement
  • Sensitivity of forward and side scatter photomultiplier tubes
  • Level of instrument baseline noise
  • Laser and optical alignment and stability
  • Stability of the fluidics system
Contents Set of 6 green-fluorescent microsphere suspensions (0.02 micron to 2.0 micron diameter).
Laser type Blue laser
Laser 488 nm
Emission 515 nm
Cat. No. F13839
  Cell sorting set up beads
Use Routine calibration of flow cytometry sorters. Beads can be used to check cell sorter settings such as drop delay and efficiency (cell loss during sorting).
Contents Fluorescent-dye infused 6 micron (+/– 10%) microspheres that have been optimized for use with either UV, blue, green/yellow and red lasers.
Laser type UV Blue laser Green/yellow laser Red laser
Laser UV 405 nm 488 nm 532/561 nm 633 nm
Emission 460 nm 515 nm 575 nm 680 nm
Fluorophore matches DAPI, Hoechst dyes FITC, Alexa Fluor 488 R-PE, tetramethylrhodamine Cy 5
Cat. No. C16505 C16508 C16509 C16507
  Rainbow Calibration Particles AlignFlow Flow Cytometry Alignment Beads
Use Routine calibration of flow cytometers
Contents Single mixture of particles featuring 8 different fluorescent intensities. Single fluorescence, laser-specific particles available in three versions (per laser excitation).
Laser type UV to red laser UV laser Blue laser Red laser
Excitation 365–650 nm 350–370 nm 488 nm 633 nm
Emission 400–680 nm 400–470 nm 515–660 nm 645–680 nm
Bead diameter 3.0–3.4 micron 2.5 micron 6.0 micron 2.5 micron 6.0 micron 2.5 micron 6.0 micron
Cat. No. A34305 A16502 A16505 A16500 A16503 A16501 A16504
  Flow Cytometry Size Calibration Kit
Use Estimate the size of cells in an experimental sample by comparing the FSC signals with those of the reference microspheres.
Contents Contains six suspensions of unstained polystyrene microspheres (1.0 micron to 15 micron diameter).
Laser type Any
Cat. No. F13838
  Flow Cytometry Sub-micron Particle Size Reference Kit
Use Check any of the following:
  • Resolution limit and dynamic range of particle size measurement
  • Sensitivity of forward and side scatter photomultiplier tubes
  • Level of instrument baseline noise
  • Laser and optical alignment and stability
  • Stability of the fluidics system
Contents Set of 6 green-fluorescent microsphere suspensions (0.02 micron to 2.0 micron diameter).
Laser type Blue laser
Laser 488 nm
Emission 515 nm
Cat. No. F13839
  Cell sorting set up beads
Use Routine calibration of flow cytometry sorters. Beads can be used to check cell sorter settings such as drop delay and efficiency (cell loss during sorting).
Contents Fluorescent-dye infused 6 micron (+/– 10%) microspheres that have been optimized for use with either UV, blue, green/yellow and red lasers.
Laser type UV Blue laser Green/yellow laser Red laser
Laser UV 405 nm 488 nm 532/561 nm 633 nm
Emission 460 nm 515 nm 575 nm 680 nm
Fluorophore matches DAPI, Hoechst dyes FITC, Alexa Fluor 488 R-PE, tetramethylrhodamine Cy 5
Cat. No. C16505 C16508 C16509 C16507

Rainbow Calibration Particles

Rainbow Calibration Particles contain a mixture of 3.0 micron particles in different fluorescent intensities. The calibration particles can be used for routine calibration of flow cytometers using wavelengths ranging from 365 nm to 650 nm.

histogram showing 8 peaks
Figure 1. Histogram analysis of particle fluorescence at 488 nm excitation and 530/30 nm emission.
scatter plot showing fluorescence at 530/30 nm and 670/14 nm
Figure 2. Particle fluorescence plotted at 488 nm excitation and 530/30 nm emission versus 633 nm excitation and 670/14 nm emission.

AlignFlow Flow Cytometry Alignment Beads

Invitrogen AlignFlow Flow Cytometry Alignment Beads are reliable references for aligning, focusing, and calibrating flow cytometers. These fluorescently stained polystyrene microspheres are highly uniform with respect to size and fluorescence intensity (Figure 3), and are designed to approximately replicate the size, emission wavelength, and intensity of biological samples. Because the dyes are contained inside the microsphere’s matrix, instead of on the bead’s surface, AlignFlow beads have excellent photochemical and physical stability, providing reliable reference signals for instrument set-up. The fluorescent dyes have been carefully selected for optimal excitation by laser sources commonly used in flow cytometry.

The AlignFlow beads are available in three versions: for 350–370 nm excitation with UV lasers, for 488 nm excitation with blue lasers, and for 633 nm excitation with red lasers. Each version is available in two bead sizes: 2.5 μm diameter and 6.0 μm diameter. See table below for product selection.

3-panel histogram


Figure 3. Invitrogen AlignFlow flow cytometry alignment beads excited at 488 nm by an argon-ion laser and monitored in three emission channels.
The broad fluorescence emission is detected in all three channels. Note the exceptionally small variation of fluorescence intensity of the beads. Contributed by Carleton Stewart, Roswell Park Cancer Institute.



Size calibration

The Invitrogen Flow Cytometry Size Calibration Kit provides a set of nonfluorescent microsphere suspensions to serve as reliable size references for cytometry users. The kit contains six suspensions of unstained polystyrene microspheres, each with a known diameter, determined by transmission electron microscopy. The size of cells in an experimental sample can be estimated by comparing the forward scatter (FSC) signals with those of the reference microspheres. The microspheres function as reproducible size markers (Figure 4) and can be intermixed with the experimental sample or used in parallel runs.

histogram analysis graph


Figure 4. Invitrogen Flow Cytometry Size Calibration Kit.
Histogram analysis of the forward scatter intensity (FSC) log channel values of the six polystyrene microsphere samples supplied in the Flow Cytometry Size Calibration Kit is shown. FSC measurements were performed on a Becton Dickinson FACScan™ flow cytometer using excitation at 488 nm.


Sub-micron particle size reference

The Invitrogen Flow Cytometry Sub-micron Particle Size Reference Kit provides a set of green-fluorescent microsphere suspensions to serve as reliable size references for flow cytometry users. The kit contains six suspensions of polystyrene microspheres, each with a known diameter as determined by transmission electron microscopy. The excitation and emission profile of all the beads is similar to Invitrogen Alexa Fluor 488 dye– or FITC-stained cells (excitation and emission maxima are 505 nm and 515 nm, respectively).

The size (or size range) of bioparticles in an experimental sample can be estimated by comparing their FSC with those of the reference microspheres (Figure 5). The microspheres in each component function as reproducible size markers and can be used individually (one size), premixed (two to six sizes), intermixed with the experimental sample, or in parallel runs. This kit can be used to verify instrument performance and to establish parameters that are suitable for analyzing sub-micron particles. For example, the kit can be used to check:

  • Resolution limit and dynamic range of particle size measurement
  • Sensitivity of forward and side scatter photomultiplier tubes
  • Level of instrument baseline noise
  • Laser and optical alignment and stability
  • Stability of the fluidics system
Flow cytometry scatter plot showing position of size standards


Figure 5. Invitrogen Flow Cytometry Sub-micron Particle Size Reference Kit.
Signals from (the kit has six particle sizes; only five were used here) five different-sized particles of the Flow Cytometry Sub-micron Particle Size Reference Kit were acquired using 488 nm excitation and a 530/30 nm band pass (BP) emission filter on the Attune Acoustic Focusing Cytometer. The diameters of the five different green-fluorescent microspheres are identified on a plot of particle fluorescence versus side scatter.


Cell sorting set up beads

Invitrogen Cell Sorting Set-Up Beads are reliable standards for the set-up and calibration of flow cytometry sorter instruments. The beads have a diameter of 6 μm (±10%), and thus approximate the size, emission wavelength, and intensity of many biological samples. Consequently, the beads can be used to check cell sorter settings such as drop delay and efficiency (cell loss during sorting). The beads can also be used to calibrate a flow cytometer’s laser source, optics, and stream flow without wasting valuable and sensitive experimental material.

Normalized emission spectra of Cell Sorting Set-up Beads


Figure 6.
Normalized emission spectra of Invitrogen Cell Sorting Set-up Beads for Blue Lasers solid line) and fluorescein-labeled cells (dashed line). The narrow emission spectrum of Cell Sorting Set-up Beads for Blue Lasers is approximately centered on the broader emission spectrum of fluorescein.

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