Photo of microspheres

What is instrument calibration?

Flow cytometry calibration is the process of adjusting the instrument to produce an accurate physical measurement. Controls include beads with an assigned value for a physical characteristic to measure or monitor the stability of the instrument.

Our control beads offer:

  • Confidence—help 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.

On demand webinar

AccuCheck ERF Reference Particles: Easy to Use Flow Cytometry Intensity Reference with Accurate NIST-Assigned Values

Use of NIST assigned ERF particles will allow accurate and reliable comparisons of flow cytometry data between instruments, laboratories and/or manufacturers. Herein, we will present why more reliable fluorescence intensity calibrators are important, features of the new AccuCheck ERF Reference Particles product and examples of how to use these new reference particles.

Flow cytometer calibration beads selection guide

AccuCheck ERF Reference Particles and ViroCheck NanoParticle Reference Kit

 AccuCheck ERF Reference ParticlesViroCheck ERF Reference Particles
UseStandardization and calibration for inter and intra instrument data comparisonStandardization and calibration for inter and intra instrument data comparison
Concentration (beads/mL)2x106 particles/mL5.0 x106 particles/mL, precise concentration assigned to 200 and 500 nm multicolor components (see vial for concentration)
Bead size3.2 μm100 nm, 200 nm, 500 nm
# peak Intensities3 (low, medium, and high)1 peak per kit component (5 components)
Excitation405–640 nm nm375–640 nm
Emission415–910 nm390–910 nm
Instrument support26 channels, 4 laser lines15 channels, 4 laser lines
NIST referenceCoumarin 30, Pacific orange, Fluorescein, Nile Red, APC, Alexa Fluor 700Alexa Fluor 405, Coumarin 30, fluorescein, Nile Red, APC, Alexa Fluor 700
Cat. No.A55950V10425
Figure 1. How to use ERF beads. Standardization of flow cytometry data is a 3-step process. The first step is to run one drop of ERF beads and collect the data in the channels required for your flow cytometry experiment. This will provide the mean fluorescent values for the 3 intensity peaks. The next step is to run a single-color control for each fluorophore used in the experiment and collect the mean fluorescent intensity. Then plot your values for a standard curve. The value collected in step 2 should fall on this curve and help provide a standardized fluorescence value.

 

AccuCheck and ViroCheck Equivalent Reference Fluorophores (ERF) beads are particles labeled with specific fluorophores to provide a fluorescent intensity reference. These beads provide a reference to accurately compare data from different types of instruments at different locations or data run on the same instrument on different days.

Benefits include:

  • Standardized ERF values assigned by NIST an independent organization
  • Values for 26 channels collected from emitted fluorescence from 415–910 nm
  • ERF values for a particular channel under a specified set of instrument conditions are comparable between instruments and over time, even when different manufacturers’ calibration beads are used
ERF Value Channel Assignment for AccuCheck ERF Particles
Laser LineEm. Filter SetCommon Fluorophore(s)NIST Ref. Fluorophore
405 nm
Violet
440/50Alexa Fluor 405 /BV421/Pacific BlueCoumarin 30
512/25BV510 / AmCyanCoumarin 30
603/48eFluor 625 / BV605 /QDot 605Pacific Orange
615/24eFluor 625 / BV605 /QDot 605Pacific Orange
670/30BV650/ QDot 655Pacific Orange
710/50eFluor 710/ BV711Pacific Orange
720/60eFluor 710/ BV711Pacific Orange
488 nm
Blue
525/35Alexa Fluor 488/ FITC/ GFP/Oregon GreenFluorescein
530/30FITC / Alexa Fluor 488/ GFPFluorescein
574/26PE / RFPNile Red
593/52PE-Texas RedNile Red
590/40PE-Texas RedNile Red
695/40PerCP-Cy5.5Nile Red
780/60PE-Cy7Nile Red
561 nm
Yellow
585/16PE / RPENile Red
620/15PE-Texas Red / mCherryNile Red
670/30PE-Cy5, PE-Alexa Fluor 647Nile Red
695/40PE-Cy5.5Nile Red
720/60PE-Cy5.5, PE-Alexa Fluor 700Nile Red
780/60PE-Cy7, PE-Alexa Fluor 750Nile Red
789/78PE-Cy7, PE-Alexa Fluor 750Nile Red
633 nm
Red
660/20APC / Alexa Fluor 647 / eFluor 660APC
670/14Alexa Fluor 647APC
670/30APCAPC
720/30Alexa Fluor 700Alexa Fluor 700
780/60APC-Cy7, Alexa Fluor 750Alexa Fluor 700
ERF Value Chanel Assignment for ViroCheck ERF Particles
Excitation laserEmission filter setNIST reference fluorophoreCommon fluors
375 nm UV405/30Alexa Fluor 405Alexa Fluor 350, Alexa Fluor 405, BUV395
450/45Alexa Fluor 405Alexa Fluor 405, Pacific Blue, BV421
450/45Coumarin 30Alexa Fluor 405, Pacific Blue, BV421
525/40Coumarin 30Pacific Orange, BV510
488 nm
Blue
530/30Fluoresceinsee previous table
574/26Nile Redsee previous table
610/20Nile RedPE- Texas Red
593/52PE-Texas RedNile Red
561 nm
Yellow
585/16PE / RPENile Red
620/15Nile Redsee previous table
610/20Nile RedPE- Texas Red
585/16Nile Redsee previous table
610/20Nile RedPE-Cy5, PE-Alexa Fluor 647
620/15Nile Redsee previous table
675/30Nile Redsee previous table
695/40Nile Redsee previous table
633 nm
Red
660/20APCsee previous table
670/14APCsee previous table
720/30APCsee previous table
720/30Alexa Fluor 700see previous table
780/60Alexa Fluor 700APC-CY7, Alexa Fluor 750
 Rainbow Calibration ParticlesAlignFlow Flow Cytometry Alignment Beads
UseRoutine calibration of flow cytometers
ContentsSingle mixture of particles featuring 8 different fluorescent intensities.Single fluorescence, laser-specific particles available in three versions (per laser excitation).
Laser typeUV to red laserUV laserBlue laserRed laser
Excitation365–650 nm350–370 nm488 nm633 nm
Emission400–680 nm400–470 nm515–660 nm645–680 nm
Bead diameter3.0–3.4 micron2.5 micron6.0 micron2.5 micron6.0 micron2.5 micron6.0 micron
Cat. No.A34305A16502A16505A16500A16503A16501A16504
 Flow Cytometry Size Calibration Kit
UseEstimate the size of cells in an experimental sample by comparing the FSC signals with those of the reference microspheres.
ContentsContains six suspensions of unstained polystyrene microspheres (1.0 micron to 15 micron diameter).
Laser typeAny
Cat. No.F13838
 Flow Cytometry Sub-micron Particle Size Reference Kit
UseCheck 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
ContentsSet of 6 green-fluorescent microsphere suspensions (0.02 micron to 2.0 micron diameter).
Laser typeBlue laser
Laser488 nm
Emission515 nm
Cat. No.F13839
 Cell sorting set up beads
UseRoutine calibration of flow cytometry sorters. Beads can be used to check cell sorter settings such as drop delay and efficiency (cell loss during sorting).
ContentsFluorescent-dye infused 6 micron (+/– 10%) microspheres that have been optimized for use with either UV, blue, green/yellow and red lasers.
Laser typeUVBlue laserGreen/yellow laserRed laser
LaserUV 405 nm488 nm532/561 nm633 nm
Emission460 nm515 nm575 nm680 nm
Fluorophore matchesDAPI, Hoechst dyesFITC, Alexa Fluor 488R-PE, tetramethylrhodamineCy 5
Cat. No.C16505C16508C16509C16507

AccuCheck ERF Reference Particles and ViroCheck NanoParticle Reference Kit

 AccuCheck ERF Reference ParticlesViroCheck ERF Reference Particles
UseStandardization and calibration for inter and intra instrument data comparisonStandardization and calibration for inter and intra instrument data comparison
Concentration (beads/mL)2x106 particles/mL5.0 x106 particles/mL, precise concentration assigned to 200 and 500 nm multicolor components (see vial for concentration)
Bead size3.2 μm100 nm, 200 nm, 500 nm
# peak Intensities3 (low, medium, and high)1 peak per kit component (5 components)
Excitation405–640 nm nm375–640 nm
Emission415–910 nm390–910 nm
Instrument support26 channels, 4 laser lines15 channels, 4 laser lines
NIST referenceCoumarin 30, Pacific orange, Fluorescein, Nile Red, APC, Alexa Fluor 700Alexa Fluor 405, Coumarin 30, fluorescein, Nile Red, APC, Alexa Fluor 700
Cat. No.A55950V10425
Figure 1. How to use ERF beads. Standardization of flow cytometry data is a 3-step process. The first step is to run one drop of ERF beads and collect the data in the channels required for your flow cytometry experiment. This will provide the mean fluorescent values for the 3 intensity peaks. The next step is to run a single-color control for each fluorophore used in the experiment and collect the mean fluorescent intensity. Then plot your values for a standard curve. The value collected in step 2 should fall on this curve and help provide a standardized fluorescence value.

 

AccuCheck and ViroCheck Equivalent Reference Fluorophores (ERF) beads are particles labeled with specific fluorophores to provide a fluorescent intensity reference. These beads provide a reference to accurately compare data from different types of instruments at different locations or data run on the same instrument on different days.

Benefits include:

  • Standardized ERF values assigned by NIST an independent organization
  • Values for 26 channels collected from emitted fluorescence from 415–910 nm
  • ERF values for a particular channel under a specified set of instrument conditions are comparable between instruments and over time, even when different manufacturers’ calibration beads are used
ERF Value Channel Assignment for AccuCheck ERF Particles
Laser LineEm. Filter SetCommon Fluorophore(s)NIST Ref. Fluorophore
405 nm
Violet
440/50Alexa Fluor 405 /BV421/Pacific BlueCoumarin 30
512/25BV510 / AmCyanCoumarin 30
603/48eFluor 625 / BV605 /QDot 605Pacific Orange
615/24eFluor 625 / BV605 /QDot 605Pacific Orange
670/30BV650/ QDot 655Pacific Orange
710/50eFluor 710/ BV711Pacific Orange
720/60eFluor 710/ BV711Pacific Orange
488 nm
Blue
525/35Alexa Fluor 488/ FITC/ GFP/Oregon GreenFluorescein
530/30FITC / Alexa Fluor 488/ GFPFluorescein
574/26PE / RFPNile Red
593/52PE-Texas RedNile Red
590/40PE-Texas RedNile Red
695/40PerCP-Cy5.5Nile Red
780/60PE-Cy7Nile Red
561 nm
Yellow
585/16PE / RPENile Red
620/15PE-Texas Red / mCherryNile Red
670/30PE-Cy5, PE-Alexa Fluor 647Nile Red
695/40PE-Cy5.5Nile Red
720/60PE-Cy5.5, PE-Alexa Fluor 700Nile Red
780/60PE-Cy7, PE-Alexa Fluor 750Nile Red
789/78PE-Cy7, PE-Alexa Fluor 750Nile Red
633 nm
Red
660/20APC / Alexa Fluor 647 / eFluor 660APC
670/14Alexa Fluor 647APC
670/30APCAPC
720/30Alexa Fluor 700Alexa Fluor 700
780/60APC-Cy7, Alexa Fluor 750Alexa Fluor 700
ERF Value Chanel Assignment for ViroCheck ERF Particles
Excitation laserEmission filter setNIST reference fluorophoreCommon fluors
375 nm UV405/30Alexa Fluor 405Alexa Fluor 350, Alexa Fluor 405, BUV395
450/45Alexa Fluor 405Alexa Fluor 405, Pacific Blue, BV421
450/45Coumarin 30Alexa Fluor 405, Pacific Blue, BV421
525/40Coumarin 30Pacific Orange, BV510
488 nm
Blue
530/30Fluoresceinsee previous table
574/26Nile Redsee previous table
610/20Nile RedPE- Texas Red
593/52PE-Texas RedNile Red
561 nm
Yellow
585/16PE / RPENile Red
620/15Nile Redsee previous table
610/20Nile RedPE- Texas Red
585/16Nile Redsee previous table
610/20Nile RedPE-Cy5, PE-Alexa Fluor 647
620/15Nile Redsee previous table
675/30Nile Redsee previous table
695/40Nile Redsee previous table
633 nm
Red
660/20APCsee previous table
670/14APCsee previous table
720/30APCsee previous table
720/30Alexa Fluor 700see previous table
780/60Alexa Fluor 700APC-CY7, Alexa Fluor 750
 Rainbow Calibration ParticlesAlignFlow Flow Cytometry Alignment Beads
UseRoutine calibration of flow cytometers
ContentsSingle mixture of particles featuring 8 different fluorescent intensities.Single fluorescence, laser-specific particles available in three versions (per laser excitation).
Laser typeUV to red laserUV laserBlue laserRed laser
Excitation365–650 nm350–370 nm488 nm633 nm
Emission400–680 nm400–470 nm515–660 nm645–680 nm
Bead diameter3.0–3.4 micron2.5 micron6.0 micron2.5 micron6.0 micron2.5 micron6.0 micron
Cat. No.A34305A16502A16505A16500A16503A16501A16504
 Flow Cytometry Size Calibration Kit
UseEstimate the size of cells in an experimental sample by comparing the FSC signals with those of the reference microspheres.
ContentsContains six suspensions of unstained polystyrene microspheres (1.0 micron to 15 micron diameter).
Laser typeAny
Cat. No.F13838
 Flow Cytometry Sub-micron Particle Size Reference Kit
UseCheck 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
ContentsSet of 6 green-fluorescent microsphere suspensions (0.02 micron to 2.0 micron diameter).
Laser typeBlue laser
Laser488 nm
Emission515 nm
Cat. No.F13839
 Cell sorting set up beads
UseRoutine calibration of flow cytometry sorters. Beads can be used to check cell sorter settings such as drop delay and efficiency (cell loss during sorting).
ContentsFluorescent-dye infused 6 micron (+/– 10%) microspheres that have been optimized for use with either UV, blue, green/yellow and red lasers.
Laser typeUVBlue laserGreen/yellow laserRed laser
LaserUV 405 nm488 nm532/561 nm633 nm
Emission460 nm515 nm575 nm680 nm
Fluorophore matchesDAPI, Hoechst dyesFITC, Alexa Fluor 488R-PE, tetramethylrhodamineCy 5
Cat. No.C16505C16508C16509C16507

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. Calibration particles indicate the sensitivity and linearity of the instrument. This helps to ensure reliable data.

Features include:

  • 7 colors
  • Multiple intensities
  • Used to calibrate sorters and analyzers
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.

Features include:

  • Use for both analyzer and sorter laser alignment
  • Provide individual colors

Read the AlignFlow product manual

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.

Features include:

  • Non-fluorescent beads
  • 1–15 µm range

Read the Flow Cytometry Size Calibration Kit product manual

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.

Features include:

  • Submicron particles from 0.1–2 µm
  • Labeled with fluorescent particles

Read the Flow Cytometry Sub-micron Particle Size Reference Kit product manual


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.

Read the Cell Sorting Set-Up Beads product manual

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.

Resources

Fluorophore and reagent selection guide for flow cytometry

Download Flow Cytometry Protocols Handbook

Spectral Flow Cytometry Fundamentals

Invitrogen eBioscience Resources—Selection guides, Best Protocols, product performance and more.

Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.

Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.

Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.

5 Steps Resources

Support

Flow Cytometry Support Center—Find technical support recommendations for your flow cytometry workflows, including tips for experimental setup and in-depth troubleshooting help.

Flow Cytometry Panel Design Support—Work with one of our technical sales specialists to discuss your experimental needs and guide you through the process.

Flow Cytometry Protocols

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