Precision Count Beads™ Protocol and Applications
Perform sample preparation and staining as usual.
Some examples of sample that may be used are:a. Cell lines, b. Whole blood, c. PBMC, d. Cells from migration assays. 
Note: Sample handling such as centrifugation, decanting, or transferring between different tubes can significantly affect cell counts and should be kept to a minimum.
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.
Add 50μl 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.
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 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.
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 should be performed as usual to identify the cell population(s) of interest.
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.
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.
