A List of Ten Applications of Flow Cytometry

  • Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. During this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Flow cytometry can be employed in cell counting, cell sorting, biomarker detection and protein engineering. The advantage of flow cytometry lies in its rapid and reliable multiparametric analysis of individual cells within heterogeneous cell populations.

    Here is a list top ten application of flow cytometry.

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    1. DNA ploidy analysis

    DNA analysis is the first and most widely used test item in flow cytometry. Since the DNA content of malignant cells is usually different from that of normal cells, there are aneuploid cells, so there are many studies to evaluate the relationship between aneuploid cells and tumor malignancy and their prognosis. DNA content detection can also provide information on the cell cycle, which is widely used in cell biology. In particular, it can indicate the course of action of cytotoxic drugs on cells. These DNA tests can also be performed simultaneously with cell surface markers, so that in mixed cell cultures, cells expressing specific markers can usually be tracked to show their growth cycle. All methods are based on the specific chemical reaction of the dye with the nucleic acid and the emission of fluorescence. Commonly used dyes are PI and DAPI.

    1. Cell viability experiment

    Using Heochest 33342 dye to specifically bind to DNA, the degree of dye binding is also different due to different cell viability, so the activity of cells can be evaluated.

    1. Count reticulocytes in peripheral blood

    Using TO dye can specifically bind to RNA, with a binding coefficient of up to 3000, so it has a very good cost performance.

    1. CD34 positive stem cell counts in peripheral blood and bone marrow collections

    It is used clinically for the determination of stem cell mathematics before bone marrow transplantation. Using the standard ISHAG protocol requires DNA or other nuclear dyes to occupy the FITC channel, PE labeled CD34 antibody, and PE-CY5 labeled CD45 antibody.

    1. Cross-lymphocyte and granulocyte toxicity test

    It is of clinical significance to detect whether there is a reaction between immunoglobulin and recipient granulocytes in the donor serum, because this reaction can cause post-transplant fever, post-transplant lung injury and immune granulocytosis. Flow cytometry can detect human immunoglobulin bound to granulocytes after incubation of whole blood samples and serum. FITC labels human immunoglobulin antibodies, PE labels granulocyte surface markers, and PE-CY5 labels HLA antibodies.

    1. Detection of platelet autoantibodies

    Platelet autoantibodies recognize human platelet antigens and cause various clinically relevant symptoms, such as neonatal autoimmune thrombocytopenia, purpura after blood transfusion, and refractory thrombocytopenia. Flow cytometry can quickly and accurately detect platelet autoantibodies. FITC labelled anti-human immunoglobulin antibody and PE label recognized platelet antibody.

    1. Transplant cross-matching

    The original cytotoxicity test is mainly used to avoid the ultra-acute removal reaction of the graft. Flow cytometry is used to monitor whether T or B cells are attacked by immunoglobulin in the recipient's serum, as a pre-test before HLA matching. Flow cytometry has become the gold standard in this field due to its high accuracy. FITC labeled anti-human immunoglobulin antibody, PE label recognized T cell CD3 or B cell CD29 antibody.

    1. Detection of early activation index CD69 of activated lymphocytes after antigen or mitotic stimulation of cells can be used to detect the effect of immunotherapy.

    Flow cytometry can use three-color analysis to monitor the activation of lymphocyte subsets: FITC labeled CD3 antibody, PE labeled CD8 antibody, and PE-CY5 labeled CD69 antibody.

    1. Detection of cell proliferation status

    Nuclear proliferation anti-PCNA, Ki67, and BrdUrd are used to measure cell proliferation and division, and are of great significance in assessing tumor prognosis. The detection of these markers is generally the same as the detection of cell surface markers. FITC labels PCNA or Ki67 or BrdUrd, and PE or (and) PE-CY5 labels cell surface markers.

    1. Chromosome analysis

    Flow cytometry staining analysis uses two specific dyes: Hoechest33258 combined with nucleotide AT; Chromomycin A3 combined with GC. Therefore, various chromosomes can be identified based on the difference of the ATCG content of the chromosomes on the two-parameter coordinates. Chromosome analysis is usually time-consuming and requires the operator's great experience, while using flow cytometry can quickly identify abnormal chromosomes, such as adding a sorting system to sort out these abnormal chromosomes for further analysis.