CytoFlex Flow Cytometer Application Notes
Figure 7. Immunophenotyping of human peripheral blood by 13-color flow cytometry. Blood was drawn from healthy donors by venipuncture into evacuated tubes containing EDTA anticoagulant. The cell surface makers were stained by adding 100 µL of blood to a cocktail of antibodies (Table 1) for 15 minutes in the dark. To lyse the red blood cells, 1 mL of VersaLyse Lysing Solution Ready-for-use (Beckman Coulter Part Number IM3648), vortexed immediately, and then incubated for 10 minutes at room temperature in the dark. After lysing, cells were stained with 2.5 µL of the IR Fixable Dye (PromoKine Catalog Number PK-PF840-3-01) and incubated for 20 minutes at room temperature in the dark. Cells were then fixed with 1 mL of 0.05% of a 1X preparation of Fixative Solution (Beckman Coulter Part Number IM3648) diluted with PBS. This sample was then analyzed on the CytoFLEX S Blue-Red-Violet-Infrared Flow Cytometer using the recommended settings from the Daily QC and applying the compensation matrix (Figure 8).
Figure 8. Compensation Matrix for 13-color Immunophenotyping Analysis. Single color stains were prepared using VersaComp Antibody Capture Beads (Beckman Coulter Part Number B22804) and antibodies (Table 1) and using CytExpert Software to run in a compensation experiment on the CytoFLEX S Blue-Red- Violet-Infrared Flow Cytometer using the recommended settings from the Daily QC. The resulting compensation matrix shows no values over 70%. Compensation in the adjacent Infrared channels is 32.22%.
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