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        Flow Cytometry


Flow Cytometry Overview

 

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.


 


 

Flow Cytometry – Primary Antibodies Recognizing Intracellular Targets

 

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:

 

 

 

 

 

Featured Primary Antibodies for Flow Cytometry Recognizing Intracellular Targets

 

 

Product Name   Catalog #   Applications Size

AKT1 PE Antibody Mouse Monoclonal 14E5.A2.B2.H9 IgG2a kappa

  200-308-I51   ELISA, WB, IHC, IF, FC   50 µg

AKT2 APC Antibody Rat Monoclonal 16G11.E8 IgG2a kappa

  200-526-E71   ELISA, WB, IHC, IF, FC   50 µg

AKT2 PE Antibody Rat Monoclonal 11F6.B2 IgG2a kappa

  200-508-J35   ELISA, WB, IHC, IF, FC   50 µg
Anti-Lysine Acetylated (AcK) (RABBIT) Antibody   600-401-939   ELISA, WB, IHC, FC   100 µg
Anti-ATG13 pS318 (RABBIT) Antibody   600-401-C49   ELISA, WB, FC   100 µg
Anti-GFP (MOUSE) Monoclonal Antibody   600-301-215   ELISA, WB, IHC, IF   1 mg
Anti-GFP (RABBIT) Antibody   600-401-215   ELISA, WB, IHC, FC   100 µg
Antibody for the detection of FLAG™ conjugated proteins (RABBIT)   600-401-383   ELISA, WB, IF, FC   250 µg
Anti-PPAR alpha (N-terminal specific) (RABBIT) Antibody   600-401-421   ELISA, WB, IHC, FC   100 µg
Anti-SMAD3 pS423 pS425 (RABBIT) Antibody   600-401-919   ELISA, WB, IHC, FC   100 µg
Anti-ATM Protein Kinase pS1981 (MOUSE) Monoclonal Antibody   200-301-500   ELISA, WB, IHC, FC   100 µg

 

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FLAG is a registered trademark of Sigma-Aldrich Co. LLC


 

Flow Cytometry – Primary Antibodies Recognizing Cell Surface Proteins

 

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:

 

 

 

 

 

Featured Antibodies for Flow Cytometry Recognizing Cell Surface Markers


Anti-Human Flow Antibodies
CD3 APC Antibody 
CD4 Fluorescein Antibody
CD8 Fluorescein Antibody

Anti-Mouse Flow Antibodies
CD3e Fluorescein Antibody
CD4 Fluorescein Antibody
CD20 Allophycocyanin Antibody

 

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Flow Cytometry – Secondary Antibodies Conjugated to FITC, PE or APC

 

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:

 

 

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Flow Cytometry – Isotype Controls


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 controls
Rat Antibody isotype controls
Human isotype control antibody

 

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