Flow cytometers are designed to perform quantitative measurements on individual cells and other particles with speed, accuracy and precision. As with all high-performance instrumentation, flow cytometers must be calibrated frequently to ensure accuracy and reliability. The stability, uniformity and reproducibility of our fluorescent microsphere products make them ideal reference standards for flow cytometry. However, because of the high variability in quantum yields of bound dyes and the heterogeneity of protein labeling, as well as problems with stoichiometry and accessibility in binding to targets, bead standards containing a known number of fluorophores per bead do not necessarily provide accurate information about the number of ligands bound to a cell.
In order to ensure accurate and reproducible quantitative results, flow cytometers should be checked at least daily for proper performance. AlignFlow and AlignFlow Plus flow cytometry alignment beads permit the calibration of a flow cytometer's laser(s), optics and stream flow without wasting valuable and sensitive experimental material. These fluorescently stained polystyrene microspheres are highly uniform with respect to both size and fluorescence intensity, and they 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 on the surface, AlignFlow beads have excellent photochemical and physical stability, providing reliable reference signals for aligning, focusing and calibrating flow cytometers. The fluorescent dyes have been carefully selected for optimal excitation by laser sources commonly used in flow cytometry.
The 2.5 µm AlignFlow flow cytometry alignment beads are available in four versions: for UV (350–370 nm) excitation (A7304), for 488 nm excitation (A7302), for 633 nm excitation (A7312) and for 630–660 nm excitation (A14835); the 6 µm AlignFlow Plus beads are available for the same four excitation-wavelength ranges: for UV (350–370 nm) excitation (A7305), for 488 nm excitation (A7303), for 633 nm excitation (A7313) and for 630–660 nm excitation (A14836). The UV light–excitable beads emit from 400 nm to 470 nm, the 488 nm light–excitable beads emit broadly from 515 nm to 660 nm (Figure 23.2.1), the 633 nm light–excitable beads emit from 645 nm to 680 nm, and the 630–660 nm light–excitable beads emit from 670 nm to 720 nm. The AlignFlow and AlignFlow Plus flow cytometry alignment beads are supplied as suspensions packaged in dropper vials for convenient dispensation.
Figure 23.2.1 AlignFlow Plus (A7303) 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.
LinearFlow Flow Cytometry Intensity Calibration Kits provide flow cytometer operators with intensity references for generating calibration curves, establishing photomultiplier settings and evaluating sample brightness. Each kit contains fluorescent microspheres in which the degree of staining has been carefully controlled to provide precisely determined intensity levels when excited in a flow cytometer (Figure 23.2.2). The microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation. The LinearFlow Flow Cytometry Intensity Calibration Kits are available in two different bead sizes (2.5 µm or 6 µm) and five different fluorescent colors covering the spectral ranges commonly encountered in flow cytometry:
- Blue (for UV excitation/430 nm emission). Available in 2.5 µm (L14812) and 6 µm (L14813) sizes; both kits contain microspheres stained at 100%, 20%, 4.0% and 0.8% relative fluorescence intensity levels.
- Green (for 488 nm excitation/515 emission). Available in 2.5 µm (L14821) and 6 µm (L14822) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels. The LinearFlow Green Flow Cytometry Low-Intensity Calibration Kits contain 2.5 µm (L14823) or 6 µm (L14824) diameter beads stained at 0.1%, 0.02%, 0.004% and 0.001% relative fluorescence intensity levels. The fluorescence from the beads with the lowest intensity level is approximately half that of the autofluorescence typically observed from unstained cells.
- Orange (for 488 nm excitation/575 nm emission). Available in 2.5 µm (L14814) and 6 µm (L14815) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels.
- Carmine (for 488 nm excitation/620 nm emission). Available in 2.5 µm (L14816) and 6 µm (L14817) sizes; both kits contain microspheres stained at 100%, 10%, 2.0%, 0.4%, 0.1% and 0.02% relative fluorescence intensity levels.
- Deep Red (for 633 nm excitation/660 nm emission). Available in 2.5 µm (L14818) and 6 µm (L14819, Figure 23.2.2) sizes; both kits contain microspheres stained at 100%, 20%, 4.0%, 0.8%, 0.2% and 0.04% relative fluorescence intensity levels.
The 365/430 nm fluorescence excitation/emission maxima of the microspheres in the LinearFlow Blue Kits provide a close spectral match to samples stained with DAPI, Hoechst 33258 or Hoechst 33342 nucleic acid stains. These kits are ideal for intensity calibration of flow cytometers equipped with UV laser excitation. The microspheres in the LinearFlow Green Kits are designed for calibrating the green (FL1) detection channel. Although the microspheres actually have an excitation maximum of ~505 nm, they are effectively excited by the 488 nm spectral line of the argon-ion laser. Their emission maximum of ~515 nm closely matches that of samples labeled with fluorescein, Oregon Green 488 or Alexa Fluor 488 dyes or with SYTOX Green nucleic acid stain. Microspheres in the LinearFlow Orange Kit are spectrally similar to phycoerythrin and tetramethylrhodamine conjugates, making this kit useful for calibrating the orange (FL2) channel. Although these microspheres actually have an excitation maximum of ~570 nm, they are effectively excited by the 488 nm spectral line of the argon-ion laser. Microspheres in the LinearFlow Carmine Kit exhibit excitation and emission spectra similar to the spectra of the propidium iodide complex with DNA or the spectra of Texas Red or Alexa Fluor 594 dyes and are suitable for calibrating the red (FL3) channel. The microspheres in the LinearFlow Carmine Kit have an excitation maximum of ~580 nm, but they can also be excited by the 488 nm spectral line of the argon-ion laser. The microspheres in the LinearFlow Deep Red Kit have maximal emission at ~660 nm, closely matching that of Alexa Fluor 647 dye, Cy5 dye and allophycocyanin, and they are useful for calibrating flow cytometers equipped with 633 nm He-Ne laser excitation. Although primarily intended for 633 nm excitation, the Deep Red LinearFlow microspheres can be adequately excited at 488 nm and will provide accurate relative intensity readings with this excitation.
Figure 23.2.2 Fluorescence intensity histogram of the six different 6 µm polystyrene bead samples supplied in the LinearFlow Deep Red Flow Cytometry Intensity Calibration Kit (L14819). Fluorescence measurements were performed with a flow cytometer using excitation at 633 nm. This histogram is a composite of two graphs; the same mixture of microspheres was sampled and analyzed using two distinct PMT voltage settings in order to cover the full intensity range.
PeakFlow flow cytometry reference beads are stained with fluorescent dyes that have been carefully selected to produce emission peaks coincident with labeled cells used in typical flow cytometry applications. The emission profiles for these standards are intentionally narrow in comparison to fluorescein-labeled cells (Figure 23.2.3). Consequently, PeakFlow beads serve as reference sources with emissions centered upon the expected fluorescence of the experimental sample. Because PeakFlow beads are highly uniform with respect to both size and fluorescence intensity, and because they approximate the size, emission wavelength and intensity of many biological samples, they can be used to calibrate a flow cytometer's laser source, optics, stream flow and cell sorting system without wasting valuable and sensitive experimental material. Furthermore, due to their narrow emission profiles, PeakFlow beads of two different fluorescent colors exhibit minimal spectral overlap, and little or no color compensation is needed when setting up for multicolor experiments.
As with all of our flow cytometry standard microspheres, PeakFlow beads are stained internally rather than on the surface. The dyes are therefore insulated from environmental interactions that could cause variable fluorescence output, resulting in excellent signal stability. These fluorescent polystyrene microspheres are supplied as suspensions packaged in dropper vials for convenient dispensation, with a choice of seven fluorescent colors and, for most products, two different sizes (Spectral characteristics of PeakFlow flow cytometry reference beads—Table 23.9).
Figure 23.2.3 Normalized emission spectra of PeakFlow Green flow cytometry reference beads (P14827, solid line) and fluorescein-labeled cells (dashed line). The narrow emission spectrum of PeakFlow beads is approximately centered on the broader emission spectrum of fluorescein.
AbC Anti-Mouse and AbC Anti-Rat/Hamster Bead Kits
The AbC Anti-Mouse Bead Kit (A10344) provides a consistent, accurate and simple-to-use technique for the setting of flow cytometry compensation when using fluorophore-conjugated mouse antibodies. The kit contains two types of specially modified polystyrene microspheres, the AbC capture beads, that bind all isotypes of mouse immunoglobulin, and the negative beads that have no antibody binding capacity. After incubation with a fluorophore-conjugated mouse antibody, the two bead components provide distinct positive and negative populations of beads that can be used to set compensation (Figure 23.2.4). Because of the consistent nature of bead scatter and high surface antibody–binding capacity, more consistent and accurate compensation settings for any combination of fluorophore-labeled mouse antibodies can be achieved. The AbC Anti-Rat/Hamster Bead Kit (A10389) provides parallel functionality for protocols using fluorophore-labeled rat or hamster antibodies. The AbC capture beads and negative beads have a diameter of approximately 6 µm (actual size for each lot is listed on the component vial). The bead suspensions are supplied in dropper vials for convenient sample application.
Figure 23.2.4 Compensation using the AbC Anti-Mouse Bead Kit (A10344). (A) R-Phycoerythrin (R-PE)–conjugated mouse anti–human CD56 antibodies (Invitrogen cat. no. MHCD56044) label the AbC capture beads for a positive signal, and negative beads provide a negative signal. (B) FITC-conjugated mouse anti–human CD3 antibodies (Invitrogen cat. no. MHCD03014) label the AbC capture beads for a positive signal, and negative beads provide a negative signal. (C) Dual-parameter plot showing gated human lymphocytes labeled with R-PE–conjugated mouse anti–human CD56 and FITC-conjugated mouse anti–human CD3 antibodies using compensation settings obtained with the AbC Anti-Mouse Bead Kit.
ArC Amine-Reactive Compensation Bead Kit
The ArC Amine Reactive Compensation Bead Kit (A10346) provides a consistent, accurate and simple-to-use technique for the setting of flow cytometry compensation when using any of the LIVE/DEAD fixable dead cell stains. The LIVE/DEAD Fixable Dead Cell Stain Kits (Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) use an amine-reactive dye labeling method to evaluate the viability of mammalian cells by flow cytometry. The ArC Amine Reactive Compensation Bead Kit includes two types of specially modified polystyrene microspheres to allow easy compensation of the LIVE/DEAD fixable stains: the ArC reactive beads (Component A), which bind any of the amine-reactive dyes, and the ArC negative beads (Component B), which have no reactivity. After incubation with any amine-reactive dye, the two kit components provide distinct positive and negative populations of beads that can be used to set compensation.
The Flow Cytometry Size Calibration Kit (F13838) provides nonfluorescent particle-size calibration standards for use in forward light scattering measurements of cell size by flow cytometry. This kit contains suspensions of six different nonfluorescent microspheres packaged in convenient dropper vials. The individual standards contain highly uniform polystyrene microspheres with nominal diameters of 1.0 µm, 2.0 µm, 4.0 µm, 6 µm, 10 µm and 15 µm.
Flow cytometry provides a rapid method for quantitating cell characteristics; however, most flow cytometers cannot directly provide the cell concentration or absolute count of cells in a sample. Absolute cell counts have been widely used in quantitating cell populations and disease progression and are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with flow cytometry population data (multiple-platform testing) or by adding an internal microsphere counting standard to the flow cytometry sample (single-platform testing). The single-platform method is preferred as it is technically less complicated and more accurate than multiple-platform testing. To facilitate this single-platform method, we offer CountBright absolute counting beads (C36950), a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths and contain a known concentration of microspheres. For absolute counts, a specific volume of the CountBright microsphere suspension is added to a specific volume of sample, such that the ratio of sample volume to microsphere volume is known. The volume of sample analyzed can be calculated from the number of microsphere events and then used with cell events to determine cell concentration. In general, at least 1000 bead events should be acquired to assure a statistically significant determination of sample volume. Sufficient reagents are provided for 100 flow cytometry assays, each using 50 µL of counting beads per test.
CountBright absolute counting beads are broadly fluorescent and can be used with either a fluorescence or scatter threshold. Fluorescence can be excited by wavelengths from UV to 635 nm; fluorescence emission can be read between 385 nm and 800 nm. The fluorescence intensity of the microspheres has been adjusted to be about 5–50 times brighter than the anticipated intensities of typically stained cells. When using a scatter threshold, the microsphere signal should be above the threshold. The microspheres can be gated by a single parameter, but a combination of parameters can be used to resolve microspheres from cells and other events.
CountBright absolute counting beads can be used with any sample type, including no-wash/lysed whole blood. The microspheres in the reagents are approximately 7 µm in diameter and have sedimentation properties similar to lymphocytes. The accuracy of cell counts based on CountBright absolute counting beads depends on sample handling and the precise delivery of the volume of beads. The CountBright absolute counting beads must be mixed well to assure a uniform suspension of microspheres. After vortexing for 30 seconds, the microsphere suspension can be pipetted by standard techniques; however, more viscous solutions such as blood require reverse pipetting for accurate volume delivery. Cell suspensions may be diluted but should be assayed without wash steps. Other sample preparation steps that can lead to cell or microsphere loss should also be avoided. For antibody protocols, CountBright absolute counting beads should be used with reagents titered for no-wash staining.