The MACS IFNg Secretion Assay Detection Kit is designed for the analysis of IFN-gamma (IFNg) secreting lymphocytes using flow cytometry. In particular, antigen specific T cells can be quantitated using this kit by measuring IFNg production in response to antigen stimulation. This system allows for the isolation of live IFNg secreting T cells in contrast to methods that require fixation and permeabilization to identify individual IFNg positive cells. The kit includes an IFNg “catch reagent” along with a fluorescently labeled IFNg detection antibody. Cells are labeled with the catch reagent (by incubating for 5 minutes on ice) in order to coat cells with an IFNg antibody. Cells are then diluted and warmed for IFNg capture for approximately 45 minutes. The second PE labeled IFNg antibody is added along with any additional staining antibodies of interest (CD4 or CD8-FITC). Cells can then be analyzed by flow cytometry.
I have used this kit to identify the antigen specificity of CD8 T cells in response to both whole antigens and peptide antigens. I typically stimulate PBMC or cytotoxic T cell cultures with antigen for 4 hours but range between 3-8 hours of stimulation depending upon the antigens. The main reason for using this kit over other methods that utilize intracellular cytokine staining is the ability to sort live IFNg secreting cells in response to antigen stimulation. By avoiding cell fixation, antigen specific T cells can be isolated and grown in culture. For example, individual IFNg positive T cells can be isolated for cloning or RNA extraction. I found the kit works fairly well in picking up IFNg secreting cells when compared to untreated cells, although the populations are not as clearly defined as with intracellular IFNg staining. Because of this, it is difficult to get purified antigen specific T cells when sorting. I have also used the enrichment and detection kit with reasonable results. This kit includes anti-PE beads that can isolate the positive staining cells using a MACS magnetic column. The sample purity can be improved by running the sample through the column multiple times.
The biggest advantage to this system is that live cells can be stained and analyzed for IFNg production. The kit is easy to use and the protocol is straightforward. Antigen stimulation times can vary, but the IFNg capture and staining process is neither time consuming nor labor intensive. In addition to IFNg staining, other antibodies can be added during the second antibody step (post capture) so that phenotypic analysis of the IFNg secreting cells is possible. A positive response is obvious when compared to unstimulated cells.
The disadvantage of this assay is the potential for cross contamination of IFNg secreting cells to non-secreters. It is extremely important to follow the protocol closely and make sure the cells are properly diluted prior to and well mixed during the cytokine capture step. Since all lymphocytes are labeled with the capture antibody, IFNg secreted by antigen specific cells could bind to cells that are non-specific. By diluting the cells as required, the cross contamination is reduced but mixing is required through out the capture incubation in order to minimize it further. The IFNg staining population is also not as well defined or as bright as seen with intracellular cytokine staining methods. These potential problems make getting a truly antigen specific pure population difficult post sorting by fluorescence activated cell sorting.
In summary, this method can work to identify IFNg secreting cells in a mixed population of cells and is fairly easy and quick to use. If live cells are needed post cytokine staining, this method is an appropriate alternative to intracellular staining protocols that require fixation of the cells in order to identify positive populations. However, cross contamination is a concern if a highly pure population is required. The company has continually improved the protocol for this product to overcome the non-specific staining issue.
Amy Hobeika, Ph.D.
Post-Doctoral Fellow
Duke University Medical Center