Flow Cytometry Cell Counting Beads

We offer multiple products for cell counting—Invitrogen CountBright Plus and CountBright Absolute Counting Beads, Invitrogen AccuCheck Counting Beads, and the Invitrogen LIVE/DEAD BacLight Bacterial Viability and Counting Kit, which reliably distinguish and quantify live and dead bacteria with the aid of a flow cytometer, even in a mixed population containing a range of bacterial types.

Cell counting beads selection guide

CountBright Plus Ready Tubes provide all the advantages of CountBright Plus Absolute Counting beads in a convenient, ready-to-use format that improves the accuracy and repeatability of cell counting, and provides convenient room temperature storage for up to 12 months. Each tube is supplied with a pre-calibrated quantity of CountBright Plus beads, removing errors associated with the handling and pipetting of beads. CountBright Plus Ready Tubes work best with Lyse/No-Wash and No-Lyse/No-Wash workflows but are flexible enough to be used in any suitable flow cytometric assay where accurate cell counts are needed. With convenient benchtop storage, CountBright Plus Ready Tubes are ready when you are.

• Compatible—loaded with a wide breadth of fluorochromes to fit a broad range of excitation and emission spectra
• Easy-to-use—simple protocols that work with multiple cell types
• Reproducible—more reliable results compared to multiple platform testing

CountBright and CountBright Plus Absolute Counting Beads 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, so 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 can be used with cell events to determine cell concentration. In general, at least 1,000 bead events should be acquired to assure a statistically significant determination of sample volume.

CountBright and CountBright Plus Absolute Counting Beads can be used with any sample type, including lysed/no-wash whole blood. The microspheres CountBright beads are approximately 7 μM in diameter and have settling properties similar to lymphocytes. In comparison, CountBright Plus beads, are approximately 4 μM in diameter and have setting properties that allow them to be on scale with T-, B- and NK cell populations when analyzing whole blood. Additionally, CountBright Plus beads are compatible with the broadest range of excitation and emission wavelengths from UV to near IR.

• Broadest excitation range—from UV to 810 nm (near IR) laser excitation
• Broadest emission range—from 385 to 900 nm (near IR) laser emission
• On scale—Beads are on-scale with most cell types, including human whole lysed blood, for ease of use
• Greater accuracy—>95% singlets for improved accuracy

Whether using CountBright or CountBright Plus Absolute Cell Counting Beads, sample preparation steps that can lead to cell or microsphere loss, such as washes, should be avoided. CountBright Plus beads can be used with either a scatter or fluorescence threshold. 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.

Explore: CountBright Absolute Counting Beads, for flow cytometry 

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• Internal control using ratio of two different color beads indicates pipetting accuracy
• Single platform is preferred over multiple platform testing, leading to consistency in results
• Easy to validate with most immunophenotyping experiments

AccuCheck Counting Beads are an efficient single platform method for absolute cell counting that combines the advantages of direct flow cytometric immunophenotyping with the use of two different fluorescent beads (A and B beads). These two fluorospheres are used as a double internal standard for blood volume calculation. A known volume of AccuCheck Counting Beads is added to the same known volume of stained blood in a lysed/no-wash technique. The beads are counted along with cells. Because the concentration of beads is known, the number of cells per microliter (the absolute count) is obtained by relating the number of cells counted to the total number of fluorescent bead events. The cell number is then multiplied by the number of total fluorospheres per unit of volume. As the AccuCheck Counting Beads system contains two different fluorospheres in a known proportion, the accuracy of the assay pipetting can be verified using the proportion of both types of beads.

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The LIVE/DEAD BacLight Bacterial Viability and Counting Kit 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 4). This kit utilizes a mixture of two nucleic acid stains—green fluorescent Invitrogen SYTO 9 dye and red-fluorescent propidium iodide—for viability determinations, and a calibrated suspension of microspheres for accurate sample volume measurements. With the appropriate mixture of the SYTO 9 and propidium iodide stains, bacteria with intact cell membranes fluorescence bright green, whereas bacteria with damaged membranes exhibit significantly less green fluorescence and often also fluoresce red. The cell type and the gram character influence the amount of red-fluorescent staining exhibited by dead bacteria. Both the SYTO 9 and propidium iodide stains are efficiently excited by the 488 nm–spectral line of the argon-ion laser found in many flow cytometers, and their nucleic acid complexes can be detected in the green and red channels, respectively; the background remains virtually nonfluorescent.

The calibrated suspension of microspheres serves as a reference standard for sample volume. The size and fluorescence of the microspheres have been carefully chosen to ensure that they will be clearly distinguishable from any stained bacteria population in a fluorescence-versus-side scatter cytogram. A bacterial culture is simply stained with the optimal mixture of SYTO 9 dye and propidium iodide, and then a fixed number of microspheres are added before analyzing the sample on a flow cytometer. Live and dead bacteria and the microspheres are all easily distinguished in a plot of fluorescence versus side scatter. The concentration of both the live bacteria and the dead bacteria can then be determined from the ratio of bacterial events to microsphere events.

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Explore: LIVE/DEAD BacLight Bacterial Viability and Counting Kit, for flow cytometry