Proliferation Assays

Determining the proliferative capacity of antigen specific cells can be a useful indicator of an immune response to a treatment.  There are several commonly used methods to measure cell proliferation.


Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) Assay

CFSE is a dye that passively diffuses into cells and binds to intracellular proteins.  Upon cell division, each daughter cell receives an equal portion of the CFSE dye, halving the fluorescence intensity of the cell as measured by flow cytometry.  On the basis of this decrease in fluorescence, the number of cell divisions occurring can be determined, and hence a measure of proliferation.

The principle of the assay in relation to immune monitoring is as follows:

  • Cells of interest are labeled with the CFSE dye

  • An appropriate stimulant is added to the cells e.g. peptides, allogeneic lymphocytes

  • Samples are stained with any other antibodies of interest

  • Cell division analysis carried out by flow cytometry

This assay can be used to monitor proliferative responses for both MHC class I and class II epitopes.  CFSE assays can also be used in combination with antibody or MHC multimer staining making a very flexible protocol providing in depth information about the cells of interest.


3H Thymidine Proliferation Assay

Another widely used proliferation assay is the 3H Thymidine uptake assay.  Cells incorporate the radiolabeled nucleotide into newly synthesized DNA.  In this way, the level of radioactivity as measured by liquid scintillation gives a relative measure of cellular proliferation.

The assay is carried out as follows:

  • Cells are incubated with an appropriate stimulant e.g peptides for several days
  • Approximately a day before the experiment ends, the cells are incubated with 3H Thymidine
  • After 18-36 hours the proliferative capacity of cells is measure by liquid scintillation relative to positive and negative controls.

This protocol can be quite time consuming, taking up to a week to complete.  It also uses radioactive reagents which requires specialist laboratory certification and specially trained users.  For this reason, 3H Thymidine assays have decreased in popularity in recent years and other proliferation assays such as CFSE have become more common.

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