Quick Guide to Proliferation Assays

Proliferation assays are some of the most commonly used measures of immune cell activation. Anne Lodge of Astarte Biologics recently appeared on a webinar detailing 14 ways to measure immune cell activation, including these five common proliferation assays. Here is a brief overview of each.

Tritiated Thymidine Incorporation

Radiolabeled thymidine is incorporated into the DNA during replication and is detected using liquid scintillation. Any uptake of thymidine is an indication of proliferation.

Pros: A clear signal-to-noise ratio makes it easy to detect a positive response.

Cons: Tritium is a radioactive substance and requires a special instrument for detection, which may be costly.

Bromodeoxyuridine Incorporation

The BrdU method works similar to the tritiated thymidine method above but can be detected using either an ELISA format or by fluorescent labeling.

Pros: Measuring proliferation using flow cytometry gives you the advantage of seeing exactly what cells incorporated the label during the experiment. Preferred over the radioactive thymidine method.

Cons: Takes additional time to fix and permeabilize the cells. Risk of losing signal if using the plate-based assay. Requires flow analysis capabilities.

Tetrazolium Dyes

Tetrazolium dyes rely on the reduction of substrate to a colored product by mitochondrial enzymes, enabling researchers to see increased conversion of the dye with increased metabolic activity.

Pros: Comes in various sorts and colors (MTT, XTT, and MTS).

Cons: Poor signal-to-noise ratio.

CFSE Dye Reduction

Carboxyfluorescein diacetate succinimidyl ester (CFSE) 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.

Pros: With each cell’s division, the individual cell fluorescence is reduced by half, which makes for a clear readout using flow cytometry. Ability to do some very elegant analyses.

Cons: Requires a larger quantity of cells to run through the cytometer and get valid numbers. Can have low throughput.

CellTiter-Glo®

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. Perhaps most useful with purified antigen-specific cells.

Pros: Very sensitive to the cell number, so you don’t need a large number of cells.

Cons: Poor signal-to-noise ratio.

This post originally appeared on the Astarte Biologics blog and has been modified for Patch.