Multiple soluble molecules in body fluids or culture supernatants can be measured by Flow Cytometry using multiplex bead-based assays. This method requires a smaller sample volume (about 25 μL) and has a higher sensitivity and speed compared to other techniques, such as ELISA.1
This application note illustrates the use of the CyAn ADP multicolor flow cytometer to acquire 11-plex samples prepared with the Bender MedSystems FlowCytomix Kit used on the culture supernatant of a CD8+ T cell line, HLA-A*0201-specific, obtained with repeated NV9 peptide (part of CMV pp65 antigen, NLVPMVATV ) pulsing and IL-2 stimulation (see Figures 2 and 3).
This kit uses two sets of size-coded beads (5,5 microns shown in R1, and 4,4 microns, shown in R2 ) composed of 5 / 6 different beads subsets, fluorescence-coded by different intensities of the Starfire Red** dye (excitation from UV to red, emission max at 675nm). Each bead is coated with a different antibody, specific for a single analyte.
The analyte concentration is measured staining the cytokine + bead complex with a secondary, biotinconjugated antibody, followed by streptavidin-PE staining.
Different PE intensities represent different analyte concentrations.
The kit includes a set of standards for each analyte. Thus, the measured intensities can easily be translated into concentration units.