Litron - Flow Cytometry Background

Background on Flow Cytometry

Flow cytometry (FCM) is a technology that allows cells in suspension to be efficiently measured at rates of up to 10,000 cells/second. As cells pass through a focused laser beam, the cell size, fluorescent dye distribution, internal structure, membrane potential, nuclear diameter may be determined, as well as, the detection DNA, enzymes, RNA, and surface antigens.

A laser beam set at a specific wavelength of light excites fluorochromes on, or inside, the cells. Once excited, these fluorochromes emit different wavelengths of light that can be detected by photomultiplier tubes (PMTs) in the flow cytometer.

Flow Cytometry and the Micronucleus Test

Traditionally, the scoring of micronuclei (MN) was performed by microscopic inspection of 1,000 to 2,000 young Red Blood Cells (reticulocytes). This was, by necessity, a very subjective, time- and labor-intensive method, giving wide standard deviations, and a greater chance for technician error.

In contrast, flow cytometers offer speeds far beyond that of microscopic inspection or image analysis. With this increased speed and objectivity, it is now possible to score 20,000 reticulocytes for micronuclei, ten times the data, in just minutes. Rather than waiting weeks or months for results, a researcher can obtain data from a typical test in just one day. This is an enormous benefit to those researchers working in competitive fields.

Litron's MicroFlow method uses a single-laser flow cytometer. The cells of interest (RETs) are labeled with FITC-conjugated antibodies directed against a cell surface antigen (transferrin receptor, CD71). Micronucleated cells are detected based on their red fluorescence from a DNA stain (propidium iodide).

Flow cytometric analysis

The results from flow cytometric analysis of a typical blood sample are shown at right. The Y axis corresponds to green fluorescence (FL1) and the X axis to red fluorescence (FL3). When analyzing micronucleated cells, the DNA (micronuclei) of each cell is stained with a red dye, while the RETs (youngest cells) are stained green.

Window 1: Mature Erythrocytes (NCEs). These cells remain unstained by the dyes, having neither red nor green fluorescence.
Window 2: Mature Micronucleated Erythrocytes (MN-NCEs). The DNA within these cells is labeled (stained) with red fluorescence.
Window 3: Young Erythrocytes (RETs). The FITC-conjugated antibody labels these cells with green fluorescence.
Window 4: Young Micronucleated Erythrocytes (MN-RETs). These cells are labeled with both red and green fluorescence. This is the population of interest for the Micronucleus Test.