Fluorescent Cell Barcoding is a technique that facilitates high-throughput multiparameter flow cytometry applications by allowing multiple target cell samples to be combined into a single tube for multicolor staining and analysis. Each cell sample is “barcoded,” using one or two covalently bound fluorescent dyes-each dye is used at up to three specific concentrations. This allows the cell samples to be distinguished by multicolor flow cytometry based on their distinct fluorescence emission wavelengths and intensities. Multiplexed barcoded cell samples can further be stained with fluorescent antibodies that are specific for CD antigens and intracellular phosphorylated signaling proteins. Thus, Fluorescent Cell Barcoding enables a high-throughput approach to determine the signaling profiles of leukocyte subsets. Analyses that could benefit from this barcoding technique include (but are not limited to) multi-donor, dose-response and/or time-course studies for cellular response modifiers (eg, drugs, cytokines, polyclonal activators, adjuvants).
The BD Phosflow™ Violet Fluorescent Cell Barcoding Kit provides reagents for successfully barcoding T cell subsets from human whole blood samples for the purpose of studying phosphorylated protein induction in the MAP Kinase and JAK/STAT signaling pathways. The kit includes two fluorescent dyes, Cell Barcoding Dye 450 (CBD450) and Cell Barcoding Dye 500 (CBD500), Fluorescent Cell Barcoding Wash Buffer, and one phosphosite-specific antibody for barcoding and immunostaining fixed and permeabilized cell samples. The barcoding dyes are used at various concentrations either separately, or in combination to barcode up to sixteen different cell samples. The Kit provides a choice of an Alexa Fluor® 647-conjugated, phosphosite-specific antibody that can be used for analysing intracellular signaling events. The following phospho-specific antibodies are available to choose from: ERK1/2 (pT202/pY204); p38 MAPK (pT180/pY182); Stat1 (pY701); Stat3 (pY705); Stat5 (pY694); or Stat6 (pY641). Each of these antibodies has been shown to stain barcoded fixed and permeabilized human peripheral blood lymphocytes.
Fluorescent antibodies specific for T-cell associated surface markers have been validated for use with the Violet Fluorescent Cell Barcoding Kit using the fixation and permeabilization procedure specified in this kit. The Lyse/Fix and Permeabilization reagents must be ordered separately. Cell samples prepared using the reagents provided in, or specified by, this kit can be analyzed using the BD FACSCanto™ II, BD LSRFortessa™ or BD™ LSR II Flow Cytometer Systems.