What are Proliferation Assays?
Proliferation assays are a category of immune cell activation tests designed to measure cell division. Proliferation assays are some of the most commonly used measures of immune cell activation. Examples include:
- Tritiated Thymidine Incorporation (3H-thymidine Incorporation)
- Bromodeoxyuridine Incorporation (BrdU)
- Tetrazolium Dyes
- CFSE Dye Reduction
Learn more about each method below.
Tritiated Thymidine Incorporation
Tritiated thymidine is the use of radiolabeled thymidine, which is incorporated into the DNA during replication and is detected using liquid scintillation. Since most immune cells aren’t dividing (they don’t make new DNA unless activated), any uptake of thymidine is an indication of proliferation. This method produces a clear signal-to-noise ratio since there isn’t any uptake to begin with, making it easy to detect a positive response.
The sample figure below shows thymidine incorporation using mouse cells in a mixed lymphocyte reaction to mimic graft rejection in vitro. When spleen cells from a C57 black six mouse were stimulated with cells from a BALB/c mouse (shown in blue and orange lines), there is a strong increase in thymidine levels.
Because thymidine levels decrease over time, you need to choose your incorporation timeline carefully. Test the data in your own lab conditions to find the ideal measurement time.
Two drawbacks to this proliferation method are that tritium is a radioactive substance and it requires a special instrument for detection, which may be cost prohibitive.
The bromodeoxyuridine (BrdU) method works similar to the tritiated thymidine method above but can be detected using either an ELISA format or by fluorescent labeling.
If using the ELISA format, add BrdU to your cell culture overnight or longer, remove the culture medium, fix the cells to adhere them to the bottom of the plate, and permeabilize them to allow the antibody to penetrate the nucleus. Then add BrdU and a detection antibody labeled with peroxidase or your label of choice.
Your data will look similar to the sample below. In this example, we used human cells and measured the amount of absorbance via the colorimetric readout to indicate the number of cells that may have incorporated the BrdU. Proliferation will vary by day depending on the concentration of antigen and the lot-to-lot variation of the antigen sample.
An alternative method for measuring the incorporation of BrdU is fluorescent labeling. If using this method, incubate your BrdU and cell culture mixture overnight, then collect the cells, stain for surface antigens, fix and permeabilize, and add a fluorescently labeled anti-BrdU. You can then measure the proliferation using flow cytometry, giving you the advantage of seeing exactly what cells incorporated the label during the experiment.
While BrdU incorporation is preferred over the radioactive thymidine method, it does take additional time to fix and permeabilize the cells. If doing the plate-based assay, there are risks of losing signal. And in the case of the fluorescent labeling, you need flow analysis capabilities.
Tetrazolium dyes rely on the reduction of substrate to a colored product by mitochondrial enzymes. Tetrazolium dyes come in various sorts and colors (some of the more common being MTT, XTT, and MTS). With increased metabolic activity, you can expect to see increased conversion of the dye.
The main drawback of using tetrazolium dyes is the poor signal-to-noise ratio. In a primary sample, there are only a small fraction of cells that will respond to a given antigen. The rest of the cells in the sample will still be alive but won’t proliferate, so you end up with a large amount of background.
CFSE Dye Reduction
Carboxyfluorescein diacetate succinimidyl ester (CFSE) labels surface proteins on cells. With each cell’s division, the individual cell fluorescence is reduced by half, which makes for a clear readout using flow cytometry.
CFSE labeling involves preparing a cell suspension, adding the fluorescent label, incubating, washing off any excess CFSE, and then culturing the cells with your chosen antigen or stimulus. The ideal culture will run for four to seven days, depending on the stimulus. You can then collect the cells and counterstain with other cell markers or viability dyes. With flow cytometry, you can do some very elegant analyses.
Refer to the example analysis below using CFSE labeling of PBMC. The top left graph is a control group of cells that were labeled and incubated. The other three graphs show the immune response to tetanus toxoid, CMV, and PHA, respectively. The CMV analysis is a good example of a strong stimulus with many cells proliferating. The PHA analysis shows the distinct rounds of cell division with the characteristic peaks to measure how far out each generation shifts.
We have also used this method to measure T regulatory cells.
The downside of the CFSE labeling method is that it requires a larger quantity of cells to run through the cytometer and get valid numbers. We have also found this method to have low throughput.
CellTiter-Glo® measures the number of cells based on ATP content and has a longer half-life, so you have a better chance to measure the luminescence before it disappears. This method is very sensitive to the cell number, so you don’t need a large number of cells.
As illustrated in the graph below, CellTiter-Glo® has the same problem as the tetrazolium dye methods in that it is hampered by the fact that there are many cells with primary responses that will give you a lot of signal, making it challenging to measure the increase in cell number. Where CellTiter-Glo® is perhaps most useful is with purified antigen-specific cells.
Ready to Learn More?
To learn more about proliferation assays and other ways to measure immune cell activation, access a recording of our full webinar, 14 Ways to Measure Immune Cell Activation.