Flow Cytometry Reference Standards - Section 23.2

Due to advances in flow cytometric instrumentation and the development of fluorescent probes, a scientist can now perform multiparameter analyses on biological samples. In order to ensure accurate and reproducible results, flow cytometers should be checked at least daily for proper performance. Molecular Probes' 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 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 aligning, focusing and calibrating flow cytometers (AlignFlow and AlignFlow Plus Flow Cytometry Alignment Beads). The fluorescent dyes have been carefully selected for optimal excitation by laser sources commonly used in flow cytometry.

The 2.5 µm AlignFlow beads are each 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 23.29), 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 23.29 AlignFlow Plus (A7303) 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 23.30). 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 (LinearFlow Blue Flow Cytometry Intensity Calibration Kits).
• 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 (LinearFlow Green Flow Cytometry Intensity Calibration Kits). 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 (LinearFlow Orange Flow Cytometry Intensity Calibration Kits).
• 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 (LinearFlow Carmine Flow Cytometry Intensity Calibration Kits).
• Deep Red (for 633 nm excitation/660 nm emission). Available in 2.5 µm (L14818) and 6 µm (L14819, Figure 23.30) sizes; both kits contain microspheres stained at 100%, 20%, 4.0%, 0.8%, 0.2% and 0.04% relative fluorescence intensity levels (LinearFlow Deep Red Flow Cytometry Intensity Calibration Kits).

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 and the Hoechst 33258 and 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 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 the fluorescein, Oregon Green 488 or Alexa Fluor 488 dyes or with the 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 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 those of the propidium iodide complex with DNA or with Texas Red or Alexa Fluor 594 conjugates and are suitable for calibrating the red 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 the Alexa Fluor 647 dye, Cy5 dye and allophycocyanin conjugates and 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 still be adequately excited at 488 nm, where they will still provide accurate relative intensity readings.

Molecular Probes' PeakFlow 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 (Spectral characteristics of PeakFlow flow cytometry reference beads - Table 23.5). The emission profiles for these standards are intentionally narrow in comparison to fluorescein-labeled cells (Figure 23.31). Consequently, PeakFlow beads serve as reference sources with emissions centered upon the expected fluorescence of the experimental sample. Furthermore, in setting up for multicolor experiments, there is minimal spectral overlap between PeakFlow beads of two different fluorescent colors, and little or no color compensation is needed (PeakFlow Flow Cytometry Reference Beads). 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 23.5).

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. 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.

Figure 23.31 Normalized emission spectra of PeakFlow 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.

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. CountBright absolute counting beads (C36950) are 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 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 settling 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.

Accurate enumeration of low numbers of bacteria in samples must be performed daily in many quality-control laboratories. To facilitate this determination by flow cytometry, Molecular Probes has developed the Bacteria Counting Kit (B7277), which provides:

• The cell-permeant, green-fluorescent SYTO BC nucleic acid stain to label bacteria
• Fluorescent polystyrene microspheres to calibrate the volume of bacterial suspension analyzed
• A detailed protocol (Bacteria Counting Kit)

The Patented SYTO BC dye, which is also available separately (S34855, Nucleic Acid Stains - Section 8.1), is a high-affinity nucleic acid stain that easily penetrates both gram-negative and gram-positive bacteria, producing an exceptionally bright green-fluorescent signal. The calibrated suspension of polystyrene microspheres contains beads that exhibit a uniform density, low-level fluorescence and optimal size to clearly separate the light scattering of the microspheres from that of most bacteria. The fluorescent microspheres in our Bacteria Counting Kit have also been recommended for the enumeration of yeast.

The Bacteria Counting Kit is particularly valuable for monitoring antibiotic sensitivity because it provides a convenient and accurate means for assessing changes in a bacterial population over time. A sample of the population is simply diluted, stained briefly with the SYTO BC dye, mixed with a fixed number of microspheres and analyzed on a flow cytometer. Signals from both the stained bacteria and the beads are easily detected in the green (fluorescein) channel of most flow cytometers and can be distinguished on a plot of forward scatter versus fluorescence (Figure 15.74). The density of the bacteria in the sample can be determined from the ratio of bacteria signals to microsphere signals in the cytogram. The Bacteria Counting Kit can be used with a variety of gram-negative and gram-positive species of bacteria and provides sufficient reagents for approximately 100 flow cytometry assays. In addition to the Bacteria Counting Kit, we offer the LIVE/DEAD BacLight Bacterial Counting and Viability Kit (L34856, Viability and Cytotoxicity Assay Kits for Diverse Cell Types - Section 15.3), which allows researchers to reliably distinguish and quantitate live and dead bacteria with the aid of a flow cytometer, even in a mixed population containing a range of bacterial types.

Figure 15.74 Flow cytometric enumeration of Bacillus cereus using Molecular Probes' Bacteria Counting Kit (B7277). Bacteria stained with the SYTO BC bacterial cell stain and mixed with known concentrations of weakly fluorescent 6 µm polystyrene microsphere standards produce a bivariate frequency distribution for forward light scatter versus green fluorescence intensity that allows the bacterial population number to be determined by reference to the clearly separated microsphere standard population, indicated by red data points.