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 .
