Flow cytometry

By Christina Towers, PhD.

Flow cytometry was developed to label and examine single cells with high throughput capacity using antibodies conjugated to fluorophores. The basic concept of flow cytometry is that a cell suspension is delivered as a single stream and is passed through a light source that uses detectors to generate data sets based off cellular properties. More specifically, the light emitted by fluorescently conjugated antibodies is channeled through selected filters to sort based off preset parameters or targets used.

Florescence activated cell sorting or Flow cytometry permits concurrent measurements of numerous florescence and light scattered events by illuming single cells or molecules in suspension as they flow through a sensing area. Distinct cells or particles could be tangibly separated corresponding to their biochemical properties and biological parameters, while the light is scattered on the molecules either in the form of forward or side scatter.

Studies on leukemias and other blood related malignancies is one of the most relevant investigational and medicinal applications of flow cytometry. In the bone marrow, normal blood cells undergo a progressive series of differentiation and branch off as myeloid, B and T cells. Hematological disorders can arise at any stage of the cell, while the differentiating cell will express a distinctive marker depending on the stage of differentiation.

FACS (Fluorescence Activated Cell Sorting) was developed by Bonner, Sweet, Hulett, Herzenberg, and others to perform flow cytometry. Flow cytometry is a powerful analytical tool useful for the characterization or phenotypic identification of different populations of cells.

Cluster of differentiation (CD) antigens are membrane proteins in nature that are predominantly expressed on the leukocyte surface. However diminutive sums of CD antigens have also been reported to be expressed on other cell types which include the endothelial, stem, and dendritic cells along with erythrocytes.

Flow cytometry is one of the powerful tools for the investigators in immunological research involved in studying various immune cells. One of the main advantages of this technique is that is capable of multi-parameter measurements that can be accomplished on a single-cell basis. As a result of the advances in the flow cytometry researchers can now decrypt the phenotypes of several cell subsets in ways that were not possible using traditional assays, such as Western/immunoblotting, microarrays and enzyme-linked immunosorbent assays.

We at Novus Biologicals have added several new products to our CD antibody database. The CD, or Cluster of Differential proteins are a family of type I transmembrane glycoproteins widely expressed in immune cell populations. These include B cells, thymocytes and peripheral T cells. Widely used as cell markers, a recent antibody study identified three CD proteins - CD44+, CD49fhi, and CD133hi – as new cell markers in an aggressive, but uncommon type of breast cancer.

Recently, we at Novus Biologicals added several embryonic stem cell marker products to our antibody catalog, validated for use in fluorescent activated cell sorting (FACS) assays. They included Cripto1, PODXL, SSEA, OCT4, Nanog, SOX2, TRA-1, TERT and GPR49/LGR5 antibodies.