TITLE: Precision Count Beads™ Protocol and Applications
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Precision Count Beads™ are designed for counting the absolute number of cells in a complex mix population and other particles by flow cytometry. Precision Count Beads™ are excited by a variety of lasers including violet (405nm), blue (488nm), yellow/green (562nm), and red (633nm).</div></div>

[STEPS]
?. [Sample Preparation]
Perform sample preparation and staining as usual. Some examples of sample that may be used are:Cell linesWhole bloodPBMCCells from migration assaysTip: Sample handling such as centrifugation, decanting, or transferring between different tubes can significantly affect cell counts and should be kept to a minimum
?. [Sample Preparation]
Vigorously vortex the Precision Count Beads™ bottle for 30-40 seconds to ensure complete mixing and break up of aggregates that may occur during storage.
?. [Sample Preparation]
Add 100µl of Precision Count Beads™ per sample. Accurate pipetting is crucial at this stage. We recommend using reverse pipetting to ensure pipetting of an accurate amount of beads.
?. [Cytometer Set Up]
Set up the FSC and SSC channels for the cells of interest as recommended or previously defined, ensuring that the FSC threshold is not too high. Precision Count Beads™ have a high SSC and low FSC profile. Thus, SSC should be adjusted in such a way that the cells are optimally visible. This can be done by acquiring a sample of Precision Count Beads™ alone and compare to the cells profile. Adjust compensation for the samples if needed.
?. [Cytometer Set Up]
Precision Count Beads™ are highly fluorescent in all channels. Make sure to adjust the voltage to optimally detect the Precision Count Beads™ in at least one fluorescent channel.
?. [Cytometer Set Up]
Acquire samples on a flow cytometer, gently vortexing every sample prior to acquisition to ensure adequate suspension of the cell and bead populations.
?. [Data Analysis]
Data analysis should be performed as usual to identify the cell population(s) of interest.
?. [Data Analysis]
In an ungated plot of the same sample, Precision Count Beads™ can be visualized using one or two fluorescent parameters and a gate set around the bead population.
?. [Data Analysis]
The bead count and cell count can be obtained using the statistics function from the software. Absolute counts can then be calculated using formulas provided in the example below.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Dilute antibody or antibodies at recommended concentration(s).
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Add 100µL of fresh EDTA blood and incubate for 15 minutes in the dark at room temperature.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Slowly vortex while adding 900µL of BioLegend’s RBC lysis/fixation (1X) solution to each tube.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Incubate for 15 minutes in the dark.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Vortex Precision Count Beads™ to ensure the beads are in a homogeneous suspension.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Add 100µL of Precision Count Beads™ using a reverse pipetting method for improved accuracy.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
Acquire samples on a flow cytometer.
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
If the volumes of the sample and the Precision Count Beads™ are the same (1:1), then the absolute cell count can be determined using the following formula:In this example, the absolute number of cells can be calculated as follows:

Precision Count Beads™ number = 2002 Cell number = 1156 Precision Count Beads per µl = 1000

Absolute Cell Count = (1156/2002) X 1000 = 577 CD3+CD4+ cells/µL
?. [Example for using Precision Count Beads™ in a lyse no wash whole blood assay]
If the relative proportion of the sample and the Precision Count Beads™ is different (not 1:1), then the absolute count can be determined as follows: