OTHER APPLICATIONS

Reporter genes.

According to the Wikipedia definition: 'A reporter gene is a gene whose product (protein) has a characteristic that allows it to be observed in the laboratory (fluorescence, detectable enzymatic activity). Reporter genes are used to visualize or measure the expression of a gene of interest. For this purpose, the reporter gene can be fused to the gene under study, or put under the control of the promoter of the latter'.

In cytometry, reporter genes are widely used for the selection of modified populations when using cell sorters. The advantage of this methodology is that it is not necessary to use an antibiotic selection. There are many different reporter genes with different spectral characteristics as shown in Figure 1 (Tounsia Aït Slimane, CHU St Antoine). It is also possible to use several reporter genes in the same experiment.

Figure 1 : Spectral characteristics of fluorescent proteins for use as reporters

Once the gene is integrated and if it is expressed, the cells become fluorescent and can be easily detected with a flow cytometer. An example of experimentation is shown in Figure 2 where the cell line that did not integrate the fluorescent gene is compared to the one that did.

Figure 2 : Comparison of a line before and after integration of a fluorescent reporter gene (GFP).


Protein quantification

Flow cytometry is a method that provides relative information about the size, morphology and intensity of a label. However, it is possible to determine with more precision the amount of proteins present on the surface of a cell or circulating by calibrating the apparatus.

For the quantification of membrane antigens, there is for example a kit of beads allowing to define a standard curve necessary for the calculation of the number of antigens present by cell (QUIFIKIT marketed by Agilent). The principle consists in using beads with a known quantity of antigens which will be used to determine the standard curve. The kit integrates, in addition to a tube with 2 types of beads (negative and high density of antibodies), five populations of beads with increasing and known densities of mouse antibodies. The presence of these antibodies will be revealed by the use of goat F(ab')2 fragments recognizing the mouse antibody present on their surface. This antibody will be used under saturating conditions to reveal the antibodies present on the beads but also the mouse antibodies used to reveal the presence of antigens on the surface of the cells tested (Figure 3). It will then be sufficient to plot the average fluorescence intensity of the studied population to determine the antigen density of the measured antigen.

Figure 3 : Principle of QUIFIKIT (© Agilent, Biocytex)


For the quantification of circulating proteins, the principle is more or less the same (Figure 4). It consists in using beads that present an antibody capable of recognizing an epitope of the protein to be analyzed as well as a fluorescent antibody capable of recognizing a second epitope of this protein but which does not interfere with the binding site of the antibody present on the surface of the beads. The protein to be quantified is thus captured by the bead and will bind to its surface in a way that is proportional to its concentration in the medium studied and the antibody will be used to reveal the presence of this protein on the surface of the bead.

As with the QUIFIKIT, by creating a standard curve, one will be able to determine with precision the concentration of the protein studied. By using beads that fluoresce according to the type of protein to be detected and by adjusting the fluorescence intensity of these beads, Becton Dickinson is able to provide kits (CBA, ©, Becton Dickinson) capable of analyzing up to 30 different proteins in the same sample at concentrations as low as 0.274 pg/liter. An example of a CBA assay is shown in Figure 5. The advantage of this type of assay is that it requires only 25 to 50 microliters of sample to analyze.


Figure 4 : Principle of CBA test (©Becton Dickinson)

Figure  5: Example of a CBA test with 6 different proteins analyzed simultaneously

Cell proliferation

Flow cytometry can study cell proliferation without relying on DNA analysis. There are molecules that diffuse freely through the cells and once inside the cells, are modified by intracellular esterases that ultimately make them highly fluorescent (e.g. CFSE). These molecules then couple to the amines of the intracellular proteins, they are not very cytotoxic and are stable up to 8 weeks.

To study cell proliferation, it is sufficient, at time zero, to put the cells in contact with these molecules and then to wash the medium and put the cells back in culture. At time zero the cells have integrated their maximum fluorescence and as at each division this fluorescence is distributed equally in the daughter cells, it is sufficient to follow the decrease in fluorescence over time to understand the number of cell divisions/proliferation (Figure 6). Dedicated calculation modules are integrated in the analysis software to determine the number of divisions.

Figure 6 : Principle of cell proliferation measurement (red time=0, green curve after several divisions)

Stem cell analysis

Stem cells are undifferentiated cells capable of generating specialized cells of all types by cell differentiation. They raise great hopes in biotherapies and are the subject of much research.

LThe first method to visualize/separate these cells by flow cytometry was based on a specific characteristic of these cells which were able to expel a dye (Hoechst 33342) from their cytoplasm towards the outside of the cell. This dye, which diffuses through the cell membrane and marks the DNA being expelled from the stem cells, showed less marking visualized by cytometry (Figure 7). By extension, these stem cell populations were called Side Population. The externalization of the dye is an active phenomenon that can be inactivated with a drug, Verapamil. The addition of Verapamil inhibits the process, so the SP cell population disappears when analyzed with Hoechst 33342. The problem with this technique was that it required the use of powerful UV lasers and was therefore not widely used.

Figure 7 : Stem cell analysis (Side Population: SP) by Hoechst 33342 exclusion

Another method is to use ALDH (aldehyde dehydrogenase) activity to isolate these stem cells. About 30 years ago, a team discovered that progenitor cells had ALDH activity. The development of a specific fluorescent substrate made it much easier to study these cells. It consists of a hydrophobic and non-toxic molecule that diffuses freely into the cells. Once in the cell, it is metabolized in ALDH cells and becomes trapped. It accumulates when the active transport systems are neutralized (mainly by a 4 degree hold). The measured fluorescence is therefore proportional to the ALDH activity. The negative control is obtained by the use of DEAB which inhibits ALDH activity (Figure 8).

Figure 8 : Labeling of ALDH activity on a cell line with the Aldefluor © kit

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