Generating dendritic cells (DCs) from PBMC CD14+ monocytes allows researchers to do a host of immunological assays. A common example of this is to examine the reactivity of a T cell mixture to a certain antigen of interest. However, prior to doing such antigen presentation assays, DCs must be properly matured in order to fully elicit a T cell response.
After generating your monocyte derived DCs (mDCs) from PBMCs, as I described in my previous post, you will have several choices on how to mature them. Two of the most common choices are to either use LPS or a monocyte maturation cocktail (MMC). LPS binds TLR4, which results in a host of downstream inflammatory genes being upregulated. Addition of IFNγ can polarize the DCs to a Th1 phenotype, while the addition of TNFα can polarize the DCs somewhat towards a Th2 phenotype. MMC, however, usually involves the addition of several molecules including TNFα, IL-6, IL-1β, and PGE2. The overall effect of this pool of molecules is to elicit a mixed Th1 and Th2 response by the DCs. Thus, the maturation method of choice is a critical choice for the researcher and may vary depending on the downstream functional assay.
Interrogating your DCs by flow cytometry is a good idea so you can be sure you have attained the cell phenotype you desire. mDCs will commonly express CD11c and CD1c and should be CD123-. Furthermore, upregulation of costimulatory molecules CD80 and CD86 and the immunoregulatory molecule CD83 and downregulation of CD14 are hallmarks of DC maturation. HLA molecules are also significantly upregulated. Remember, these molecules are not just cell markers, but have important functional relevance. The upregulation of costimulatory molecules is critical for the activation of T cells and the upregulation of surface HLA molecules is a reflection is the enhance antigen presentation capability of a mature DC.
Running your DCs on the flow cytometer will require a few special tweaks on your normal cytometer settings. The first thing you will notice is that the DCs are rather massive and irregular shaped cells. You will therefore likely need to significantly decrease both your forward scatter and side scatter to locate them on your dot plot. Secondly you will want to significantly decrease the voltages for all the channels detecting fluorchromes on your DC activation markers. These activation markers are expressed at such a high level on the DCs, that they are incredibly bright. A third issue is the high level of auto-fluorescence on DCs. It is always a good idea to have some extra DCs you can run while setting up your voltages to make sure your CD marker fluorochromes are all on scale. Be sure to use the activated sample of DCs for this! Once you have verified your settings will work, you can then proceed to normal compensation set up.
Once your cytometer settings are established your cells are ready to assay. It is a good idea to have a sample of DCs that you did not stimulate as a control to compare your matured DCs to. In my experience the best way to compare markers, such as CD83, CD86, HLA-ABC, and HLA-DR, is by using histogram overlays. Their upregulation can often be a slight shift in fluorescent intensity, which you can readout by graphing Median Fluorescent Intensity (MFI). Of course be sure that you have titered your antibodies appropriately and use isotype controls when you can. Also keep in mind that comparing MFI readouts between different assay days, different stains, and different experiments is virtually impossible. Try to group your assays whenever possible, but if not, fold change in MFI is a useful, though not ideal, calculation for comparing these sorts of data.
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Colt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.