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, 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 two products for cell counting—Invitrogen™ CountBright™ Absolute Counting Beads and Invitrogen™ AccuCheck Counting Beads and also one product, Invitrogen™ LIVE/DEAD™ BacLight™ Bacterial Viability and Counting Kit, for reliably distinguishing and quantitating live and dead bacteria with the aid of a flow cytometer, even in a mixed population containing a range of bacterial types.

Which counting product is right for your experiment?

  CountBright Absolute Cell Counting Beads AccuCheck Counting Beads LIVE/DEAD BacLight Bacterial Viability and Counting Kit
Sample type Whole blood Any type Bacterial sample
Bead Size 7 µM Bead A: 6.40 µM
Bead B: 6.36 µM
6 µM
Excitation (nm) UV to 635 Bead A: 488
Bead B: 635
Emission max (nm) 385 to 800 Bead A: 575–585
Bead B: 660–680
Parameter measured Number of cells Number of cells and accuracy of pipetting Number of events in the live bacteria region (or dead bacteria region) and number of events in the bead region
Cat. No.
C36950 PCB100 L34856

CountBright Absolute Counting Beads

  • Compatible with every commercially available instrument because they are loaded with a wide breadth of fluorochromes
  • Easy-to-use protocol that works with multiple cell types including lysed/no-wash whole blood
  • More reliable than multiple platform testing

CountBright Absolute Counting Beads are a calibrated suspension of microspheres that are brightly fluorescent across a wide range of excitation and emission wavelengths (UV to 635 nm excitation and 385 to 800 nm emission) 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 (Figure 1). In general, at least 1,000 bead events should be acquired to assure a statistically significant determination of sample volume.

CountBright Absolute Counting Beads can be used with any sample type, including lysed/no-wash whole blood. The microspheres in the reagents are approximately 7 μm in diameter and have settling properties similar to lymphocytes. Sample preparation steps that can lead to cell or microsphere loss, such as washes, should be avoided. CountBright 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 1. Invitrogen™ CountBright™ Absolute Counting Beads.
A mixture of live and heat-killed Jurkat cells were treated with reagents in the InvitrogenLIVE/DEAD™ Viability/Cytotoxicity Kit. InvitrogenCountBright™ Absolute Counting Beads were added to the sample, which was then analyzed by flow cytometry using 488 nm excitation. Calcein fluorescence was collected with a 530/30 nm bandpass filter and ethidium homodimer-1 (EthD-1) fluorescence was collected with a 610 nm longpass filter. The data show clear separation of live and dead cells, as well as separation of the counting beads.

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.

LIVE/DEAD BacLight Bacterial Viability and Counting Kit

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 2). 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.

scatter plots shoing quantififaciton of cell populations

Figure 2. 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).