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Flow Cytometry Cell Counting Beads
Flow cytometry provides a rapid method to quantify 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 quantifying cell populations and disease progression research, including in studies of stem cells. Absolute cell counts are generally obtained either by combining a separate cell concentration determination from a hematology analyzer with flow cytometric population data (multiple platform testing) or by adding an internal microsphere counting standard to the flow cytometric sample (single platform testing).
The single platform method is preferred as it is technically less complicated and it avoids interlaboratory variation and underestimations, making it more accurate than multiple platform testing.
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.
| CountBright Plus Ready Tubes | CountBright Plus Absolute Counting Beads | CountBright Absolute Counting Beads | AccuCheck Counting Beads | |
|---|---|---|---|---|
| Sample type | Any type, including whole blood and lysed/no-wash whole blood | Any type | ||
| Bead size | 4 µM | 4 µM | 7 µM | Bead A: 6.40 µM Bead B: 6.36 µM |
| Excitation (nm) | UV to 808 | UV to 808 | UV to 635 | Bead A: 488 Bead B: 635 |
| Emission (nm) | 385 to 860 | 385 to 860 | 385 to 800 | Bead A: 575–585 Bead B: 660–680 |
| Parameter measured | Number of cells | Number of cells | Number of cells | Number of cells and accuracy of pipetting |
| Cat. No. | C40000 | C36995 | C36950 | PCB100 |
| LIVE/DEAD BacLight Bacterial Viability and Counting Kit | |
|---|---|
| Sample type | Bacterial sample |
| Bead Size | 6 µM |
| Excitation (nm) | Non-fluorescent |
| Emission max (nm) | NA |
| Parameter measured | Number of events in the live bacteria region (or dead bacteria region) and number of events in the bead region |
| Cat. No. | L34856 |
| CountBright Plus Ready Tubes | CountBright Plus Absolute Counting Beads | CountBright Absolute Counting Beads | AccuCheck Counting Beads | |
|---|---|---|---|---|
| Sample type | Any type, including whole blood and lysed/no-wash whole blood | Any type | ||
| Bead size | 4 µM | 4 µM | 7 µM | Bead A: 6.40 µM Bead B: 6.36 µM |
| Excitation (nm) | UV to 808 | UV to 808 | UV to 635 | Bead A: 488 Bead B: 635 |
| Emission (nm) | 385 to 860 | 385 to 860 | 385 to 800 | Bead A: 575–585 Bead B: 660–680 |
| Parameter measured | Number of cells | Number of cells | Number of cells | Number of cells and accuracy of pipetting |
| Cat. No. | C40000 | C36995 | C36950 | PCB100 |
| LIVE/DEAD BacLight Bacterial Viability and Counting Kit | |
|---|---|
| Sample type | Bacterial sample |
| Bead Size | 6 µM |
| Excitation (nm) | Non-fluorescent |
| Emission max (nm) | NA |
| Parameter measured | Number of events in the live bacteria region (or dead bacteria region) and number of events in the bead region |
| Cat. No. | L34856 |
Cell counting beads
CountBright Plus Ready Tubes
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.
Figure 1. CountBright Plus beads and CountBright Plus Ready Tubes, spanning UV-NIR emission, show broader range of fluorophores. CountBright Plus Beads (blue) and CountBright Plus Ready Tubes (red) can be detected simultaneously with CD4-FITC stained cells in lysed whole blood when excited with a blue laser (488 nm) with a 520/20 emission filter.
CountBright and CountBright Plus Absolute Counting Beads
- 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.
CountBright Plus Absolute Counting Beads offer:
- 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.
Figure 2. Compared to CountBright beads, CountBright Plus beads can be used with a broader range of fluorophores. Unlike CountBright Beads, the CountBright Plus Beads (red) can be detected simultaneously with CD19 APC-eFluor780 stained cells (pink) in lysed whole blood when excited with a IR laser (808nm) with a 840/20 emission filter.
Figure 3. Get greater laser compatibility from UV to NIR with CountBright Plus Absolute Counting Beads. The cell counting beads can be detected in almost any fluorescence channel from 385 to 860, whereas the original CountBright Absolute Couting beads could not be detected in the near IR channel (G emission spectra above).
Figure 4. CountBright Plus Absolute Counting Beads are simultaneously on scale with T-, B- and NK cell populations when analyzing whole blood. Normal human peripheral blood was stained with Anti-Human CD3 Alexa Fluor 488 and Anti-Human CD19 PE-Cy7, then lysed with 1-Step™ Fix/Lyse Solution. CountBright Plus Absolute Counting Beads (boxed, purple) were added to the sample prior to analysis on a flow cytometer.
AccuCheck Counting Beads
- 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.
Bacterial counting beads
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.
Figure 5. Analysis of bacterial cultures using the Invitrogen LIVE/DEAD BacLight Bacterial Viability and Bacteria Counting Kit. Suspensions of live (untreated) and dead (alcohol-treated) Staphylococcus aureus(panels A and C) and Escherichia coli(panels B and D) were stained with Invitrogen SYTO 9 nucleic acid stain and propidium iodide and then analyzed by flow cytometry according to the kit protocol. The green or red fluorescence versus side scatter cytogram (panel A or B) was used to gate the bacterial population and the bead population (left and right boxes, respectively). Events in the bacteria region of each cytogram are also displayed in red fluorescence versus green fluorescence cytograms (panels C and D). Live and dead bacteria/mL can be calculated from either the fluorescence versus side scatter cytogram or the green fluorescence versus red fluorescence cytogram, depending on which one shows the best separation of the live and dead populations. The position of the live and dead populations in these cytograms may be dependent on cell type and gram character. Some samples may exhibit events that fall outside the defined regions and should be evaluated appropriately (e.g., see panel D).
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.