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).
Example for using Precision Count Beads™ in a lyse no wash whole blood assay
1. Dilute antibody or antibodies at recommended concentration(s).
2. Add 100µL of fresh EDTA blood and incubate for 15 minutes in the dark at room temperature.
3. Slowly vortex while adding 900µL of BioLegend’s RBC lysis/fixation (1X) solution to each tube.
4. Incubate for 15 minutes in the dark.
5. Vortex Precision Count Beads™ to ensure the beads are in a homogeneous suspension.
6. Add 100µL of Precision Count Beads™ using a reverse pipetting method for improved accuracy.
7. Acquire samples on a flow cytometer.
Figure 1. It is easier to visualize Precision Count Beads™ using at least one fluorescent channel as a parameter.
8. 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:
Figure 2. Human peripheral blood lymphocytes were stained with CD3 FITC/CD4 PE/CD45 PerCP cocktail (BioLegend Cat #368302) using a lyse/no wash method. Precision Count Beads™ were used as recommended. Bead count and CD3+CD4+ cell count was determined by gating on beads and cells as depicted in Figure 2. Cells were gated on lymphocytes, based on their FSC vs SSC profile, and CD45+.
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
9. 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: