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Flow Cytometer Calibration and Size Reference Beads
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.
Flow cytometer calibration beads selection guide
| AccuCheck ERF Reference Particles | ViroCheck ERF Reference Particles | |
|---|---|---|
| Use | Standardization and calibration for inter and intra instrument data comparison | Standardization and calibration for inter and intra instrument data comparison |
| Concentration (beads/mL) | 2x106 particles/mL | 5.0x106 particles/mL, precise concentration assigned to 200 and 500 nm multicolor components (see vial for concentration) |
| Bead size | 3.2 μm | 100 nm, 200 nm, 500 nm |
| # peak Intensities | 3 (low, medium, and high) | 1 peak per kit component (5 components) |
| Excitation | 405–640 nm | 375–640 nm |
| Emission | 415–910 nm | 390–910 nm |
| Instrument support | 26 channels, 4 laser lines | 15 channels, 4 laser lines |
| NIST reference | Coumarin 30, Pacific orange, Fluorescein, Nile Red, APC, Alexa Fluor 700 | Alexa Fluor 405, Coumarin 30, fluorescein, Nile Red, APC, Alexa Fluor 700 |
| Cat. No. | A55950 | V10425 |
| 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 6 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:
|
| 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. | C16506 | C16508 | C16509 | C16507 |
| AccuCheck ERF Reference Particles | ViroCheck ERF Reference Particles | |
|---|---|---|
| Use | Standardization and calibration for inter and intra instrument data comparison | Standardization and calibration for inter and intra instrument data comparison |
| Concentration (beads/mL) | 2x106 particles/mL | 5.0x106 particles/mL, precise concentration assigned to 200 and 500 nm multicolor components (see vial for concentration) |
| Bead size | 3.2 μm | 100 nm, 200 nm, 500 nm |
| # peak Intensities | 3 (low, medium, and high) | 1 peak per kit component (5 components) |
| Excitation | 405–640 nm | 375–640 nm |
| Emission | 415–910 nm | 390–910 nm |
| Instrument support | 26 channels, 4 laser lines | 15 channels, 4 laser lines |
| NIST reference | Coumarin 30, Pacific orange, Fluorescein, Nile Red, APC, Alexa Fluor 700 | Alexa Fluor 405, Coumarin 30, fluorescein, Nile Red, APC, Alexa Fluor 700 |
| Cat. No. | A55950 | V10425 |
| 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 6 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:
|
| 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. | C16506 | C16508 | C16509 | C16507 |
Fluorescent calibration beads
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 increase the accuracy of compared 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
Click image to enlarge
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.
| Laser Line | Em. Filter Set | Common Fluorophore(s) | NIST Ref. Fluorophore |
| 405 nm Violet | 440/50 | Alexa Fluor 405 /BV421/Pacific Blue | Coumarin 30 |
| 512/25 | BV510 / AmCyan | Coumarin 30 | |
| 603/48 | eFluor 625 / BV605 /QDot 605 | Pacific Orange | |
| 615/24 | eFluor 625 / BV605 /QDot 605 | Pacific Orange | |
| 670/30 | BV650/ QDot 655 | Pacific Orange | |
| 710/50 | eFluor 710/ BV711 | Pacific Orange | |
| 720/60 | eFluor 710/ BV711 | Pacific Orange | |
| 488 nm Blue | 525/35 | Alexa Fluor 488/ FITC/ GFP/Oregon Green | Fluorescein |
| 530/30 | FITC / Alexa Fluor 488/ GFP | Fluorescein | |
| 574/26 | PE / RFP | Nile Red | |
| 593/52 | PE-Texas Red | Nile Red | |
| 590/40 | PE-Texas Red | Nile Red | |
| 695/40 | PerCP-Cy5.5 | Nile Red | |
| 780/60 | PE-Cy7 | Nile Red | |
| 561 nm Yellow | 585/16 | PE / RPE | Nile Red |
| 620/15 | PE-Texas Red / mCherry | Nile Red | |
| 670/30 | PE-Cy5, PE-Alexa Fluor 647 | Nile Red | |
| 695/40 | PE-Cy5.5 | Nile Red | |
| 720/60 | PE-Cy5.5, PE-Alexa Fluor 700 | Nile Red | |
| 780/60 | PE-Cy7, PE-Alexa Fluor 750 | Nile Red | |
| 789/78 | PE-Cy7, PE-Alexa Fluor 750 | Nile Red | |
| 633 nm Red | 660/20 | APC / Alexa Fluor 647 / eFluor 660 | APC |
| 670/14 | Alexa Fluor 647 | APC | |
| 670/30 | APC | APC | |
| 720/30 | Alexa Fluor 700 | Alexa Fluor 700 | |
| 780/60 | APC-Cy7, Alexa Fluor 750 | Alexa Fluor 700 |
| Excitation laser | Emission filter set | NIST reference fluorophore | Common fluors |
| 375 nm UV | 405/30 | Alexa Fluor 405 | Alexa Fluor 350, Alexa Fluor 405, BUV395 |
| 450/45 | Alexa Fluor 405 | Alexa Fluor 405, Pacific Blue, BV421 | |
| 450/45 | Coumarin 30 | Alexa Fluor 405, Pacific Blue, BV421 | |
| 525/40 | Coumarin 30 | Pacific Orange, BV510 | |
| 488 nm Blue | 530/30 | Fluorescein | see previous table |
| 574/26 | Nile Red | see previous table | |
| 610/20 | Nile Red | PE- Texas Red | |
| 593/52 | PE-Texas Red | Nile Red | |
| 561 nm Yellow | 585/16 | PE / RPE | Nile Red |
| 620/15 | Nile Red | see previous table | |
| 610/20 | Nile Red | PE- Texas Red | |
| 585/16 | Nile Red | see previous table | |
| 610/20 | Nile Red | PE-Cy5, PE-Alexa Fluor 647 | |
| 620/15 | Nile Red | see previous table | |
| 675/30 | Nile Red | see previous table | |
| 695/40 | Nile Red | see previous table | |
| 633 nm Red | 660/20 | APC | see previous table |
| 670/14 | APC | see previous table | |
| 720/30 | APC | see previous table | |
| 720/30 | Alexa Fluor 700 | see previous table | |
| 780/60 | Alexa Fluor 700 | APC-CY7, Alexa Fluor 750 |
Instrument calibration beads
Rainbow Calibration Particles contain a mixture of 3 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
Figure 2. Histogram analysis of particle fluorescence at 488 nm excitation and 530/30 nm emission.
Figure 3. Particle fluorescence plotted at 488 nm excitation and 530/30 nm emission versus 633 nm excitation and 670/14 nm emission.
Read the Rainbow Calibration Particles manual
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
Figure 4. 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 reference beads
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 6 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
Figure 5. 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 reference beads
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 6 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 (1 size), premixed (2–6 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
Figure 6. 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 calibration 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.
Figure 7.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.
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.
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