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Flow cytometry is a technology that allows rapid and quantitative multi-parameter assays on single living (or dead) cells by measuring visible and fluorescent light emission signals. Furthermore, by analyzing the expression of intracellular and cell surface molecules using fluorophore-labeled antibodies, flow cytometry provides quantitative information of antigenic, biochemical and biophysical characteristics of individual cells. This makes possible the characterization and identification of different cell types in heterogeneous populations and the determination of purity of isolated subpopulations. In more advanced applications such as cell sorting, flow cytometers can also integrate the analysis results to a fluidic diversion system that enables sorting and collection of cells based on desired parameters. The accuracy of flow cytometry analysis greatly depends on the use of suitable antibodies and optimized staining methods. Antibody titration and reactivity against the target antigen should be carefully determined and appropriate isotype controls should always be included in experimental designs. Rockland provides scientists with antibodies and supporting reagents tested and qualified for flow cytometry analysis including primary antibodies that recognize intracellular or cell surface cellular targets, secondary antibodies for flow cytometry and isotype control proteins.
Staining of intracellular targets - often activated (i.e. phosphorylated) enzymes in key cell signaling pathways - for analysis by flow cytometry depend on the ability to successfully fix and permeabilize the cell membrane so that labeled antibodies can enter the cell (permeabilize) and interact with their target antigen. Rockland recommends our primary antibodies for flow cytometry that recognize post-translational modifications (ptms) on key cell signaling pathway components involved in cancer, nuclear signaling, developmental biology, epigenetics, immunology, microbiology, neuroscience, signal transduction and stem cells:
AKT1 Antibody APC Conjugated200-326-J34
AKT1 Antibody PE Conjugated200-308-J34
Conjugation of fluorescent molecules to antibodies constitutes a valuable tool for the detection of cell surface markers and other extracellular antigens by flow cytometry. A common approach involves the multicolor staining of individual antigens to analyze the expression of proteins on the cell surface only, or in combination with the analysis of intracellular targets. Rockland offers a wide variety of antibodies specific for human and murine cell surface proteins such as receptors for growth factors and cytokines (EGFR, VEGFR, PDGFR etc.), transporters, channels proteins and CD (cluster of differentiation) markers validated for immunophenotyping via flow cytometry:
CD3 Fluorescein Antibody 200-302-M62
CD47 Phycoerythrin Antibody200-308-M68
CD8 APC Antibody200-326-M64
Anti-Human Flow AntibodiesCD3 APC Antibody CD4 Fluorescein Antibody CD8 Fluorescein Antibody Anti-Mouse Flow AntibodiesCD3e Fluorescein Antibody CD4 Fluorescein Antibody CD20 Allophycocyanin Antibody
Cell staining for flow cytometry with a non-labeled antibody that recognizes the target antigen requires treatment with a fluorophore-labeled secondary antibody, which exhibits specificity for the first antibody. Alternatively, the primary antibody can be conjugated to biotin and following binding to the target antigen, it is recognized using streptavidin conjugated to a fluorophore. Rockland recommends the following secondary antibodies for flow cytometry:
Robust data from flow cytometry greatly depends on the specificity of the antibody used. A reliable method for discrimination of background and authentic fluorescent signal is the use of a negative control antibody of the same isotype as the fluorophore-labeled primary or secondary antibody employed. Non-specific staining can be attenuated using Rockland’s isotype controls including:Mouse Antibody isotype controlsRat Antibody isotype controlsHuman isotype control antibody
Rockland Immunochemicals Inc.Gilbertsville, PA 19525E-mail: email@example.comPhone: 800.656.7625