Many immune responses activate specific cell subsets, and researchers often need to monitor the activation status of cells when evaluating a novel drug or treatment. Evidence of activation differs between cell types and may include cell proliferation, cytokine production, or modulation in cell surface marker expression. Activation assays have existed for decades, and early methods for measuring cell proliferation relied on the use of radioactive thymidine.[1] ELISPOT assays have also been a historically popular technique for measuring cytokine production by immune cells[2], but flow cytometry-based assays have now become the prevailing method for monitoring all aspects of cell activation.
Although many non-flow cytometry-based activation assays have been used for both basic and clinical research, newer immune cell activation by flow cytometry can be adapted to any cell type and can be done concurrently with other flow cytometry assays as long as the flow cytometer can accommodate a staining panel with all of these components. Certain assays may require the use of fresh and non-cryopreserved specimens. Other assays can use previously cryopreserved samples, which can be ideal for long-term clinical studies in which samples are collected at several timepoints and assayed together at a later date.
Consider incorporating one of these flow cytometry-based activation assays into your future research plans.
1. B cell or T cell:
B cells require engagement of two cell surface receptors for activation. The B cell receptor, which is a membrane-bound antibody, must bind to its cognate antigen, and secondary B cell signals may include engagement of CD40 or MHC II-peptide complexes with CD40L and the T cell receptor, respectively. Activated B cells express different cell surface markers, such as B220 and CD3, and intracellular cytokines, and different cell surface or intracellular expression patterns (immunophenotypes) are associated with various stages of differentiation into plasma cells, memory cells, or other B cell subsets.[3] Consider how you plan to activate B cells in vitro and which subsets you want to examine as you design a B cell activation experiment.
2. T cell activation:
T cells also need multiple signals for activation, including engagement of both the T cell receptor (TCR) and costimulatory molecules such as CTLA-4 and PD-1[4]. Different T cell subsets are characterized by functional and phenotypic features, and include CD4+ T helper cells, cytotoxic T cells, and regulatory T cells, as well as many others. Determine which subsets you need to examine and what markers are associated with activation as you design in vitro activation assays.
3. Proliferation:
Flow cytometry-based immune cell proliferation assays are quickly becoming the standard approach for measuring proliferation. Two primary methods are in use: a fluorescent dye-based assay using carboxyfluorescein diacetate succinimidyl ester (CFSE) or similar fluorescent stains are dyes that can be incorporated intracellularly into living cells and are diluted during subsequent cell divisions. Cell proliferation can also be measured by quantifying DNA synthesis during the S phase of the cell cycle using a synthetic analog of thymidine called 5-bromo-2’-deoxyuridine (BrdU). BrdU is added in cell culture and incorporation into DNA is measured using intracellular cytokine staining with secondary antibodies against BrdU. CFSE or BrdU measurements can be part of a staining panel as long as your cytometer can detect all of the dyes being used.
4. Macrophage and Dendritic Cell (DCs) Activation:
Macrophages and dendritic cells are phagocytic cells that are part of the first line of defense against infection. These cells can take up and destroy a wide array of pathogens and present antigens from these invaders to T cells and B cells to initiate adaptive immune responses. Similar to B cells and T cells, subsets of macrophages and DCs differentiate upon activation. Flow cytometry panels have been developed to immunophenotype these subsets concurrently, and the flow cytometry platform is ideal for quantifying DCs, as these cells are typically rare and can be more difficult to measure in other in vitro assays.[5]
Flow cytometry is a powerful technique for assessing cell activation across immune cell subsets. Numerous activation and staining protocols exist for each cell type, so consider discussing your protocol options with an expert scientist working in this area, and never underestimate the value of doing pilot experiments and optimizing protocols.
Learn More About Flow Cytometry
[1] Gillis S, Ferm MM, Ou W, Smith KA. T cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol. 1978 Jun;120(6):2027-32.
[2] Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A (1983). "A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells". J Immunol Methods. 65 (1–2): 109–121
[3] Kaminski DA, Wei C, Rosenberg AF, Lee FE-H, Sanz I. Multiparameter Flow Cytometry and Bioanalytics for B Cell Profiling in Systemic Lupus Erythematosus. Methods in molecular biology (Clifton, NJ). 2012;900:109-134.
[4] Ott PA, Hodi FS, Robert C. CTLA-4 and PD-1/PD-L1 Blockade: New Immunotherapeutic Modalities with Durable Clinical Benefit in Melanoma Patients 2013. Clin Cancer Res. (19) 5300-5309.
[5] Misharin AV, Morales-Nebreda L, Mutlu GM, Budinger GRS, Perlman H. Flow Cytometric Analysis of Macrophages and Dendritic Cell Subsets in the Mouse Lung. American Journal of Respiratory Cell and Molecular Biology. 2013;49(4):503-510.
