Although assessing cell cycle distribution by DNA content will show the number of cells in each cell cycle compartment, it does not give a true indication of cellular proliferation. Flow cytometric assays that further assess cellular proliferation are based primarily on two characteristics: the division of cells and the replication of DNA.
The rate at which cells are dividing is a direct measure of cellular proliferation. When a cell divides the daughter cells each comprise half of the parantel cell. By labelling the parental cell with a fluorescent dye we are able to trace the division of cells by the dilution of that dye during the first and susequent cell divisions.
The most commonly used fluorescent dyes are those that label abundant cellular components such as lipid (e.g. PKH-26, DiO) or protein (e.g. CFSE, CMTMR). Cells are loaded with high levels of the dye before culture/treatment, and dilution of the dye can be tracked by flow cytomtry as cells divide over time.
The figure below shows a schematic of cell division and a histogram overlay showing unstimulated cytotoxic T cells (shaded grey) and different levels of proliferation induced by two different treatments (shown in blue and red). The first, second, and third divisions can clearly be seen in red. The beauty of this technique is that when combined with the multiparameter capabilities of flow cytometry it enables cellular division to be easily tracked in hetergenous populations of cells.
Incorporation of thymidine analogues
One of the first events to occur upon the initiation of cellular division is the replication of DNA. The incorporation of thymidine analogues, such as bromodeoxyuridine (BrDu) and 5-ethanyl 2-deoxyuridine (EdU), into DNA during DNA synthesis can be measured using flow cytometry.
Once incorporated into the DNA, BrDu can be detected using an anti-BrDu antibody and EdU by ClickIT technology (from Invitrogen). Both assays can be combined with DNA content to further determine cell cycle distribution.
In order for BrDu to be detectable by an anti-BrDu atibody the DNA has to first be denatured to allow the antibody access to the BrDu. This can be achieved by either treatment with HCl or DNase. HCl is very harsh to the cells making it incompatible with the detection of other antigens using immunofluorescence techniques, and usually results in a lot of cell loss during sample preparation.
DNase treatment is a much gentler mode of denaturing DNA, but it can be tricky to optimise and DNA profiles may be affected due to excessive fragmentation of DNA. A major drawback of both of these techniques is that neither is compatible with the detection of fluorescent reporter proteins.
Cellular permiablisation is necesssary to allow access of the anti-BrDu antibody into the cell but this also allows fluorescent proteins to leave the cell, making it difficult or impossible to distinguish cells that contained the reporter protein from those that do not.
The EdU Click IT assay from Invitrogen detects EdU incoportaed into the DNA without the need for denaturation of DNA. This makes the assay fully compatible with the detection of other cellular antigens (both extracellular and intracellular) and fluorescent reporter proteins. However, the assay is considerably more expensive to perform than the BrDu assay.
Irrespective of which mode of detection is used, the flow cytometric data produced is presented in the same way (see figure below). DNA content (measured by a dye labelling total DNA) shows cell cycle distribution, and BrDu/EdU content shows the cells incorporating these analogues during exposure to the reagent. Not only can the number of proliferating cells be determined but also the rate at which they are proliferating, by quantifying the amount of BrDu/EdU that has been incorporated.