Exploring cultured and co-cultured ELISpot methods
Published: October 1, 2024
Updated: October 3, 2024
ELISpot and FluoroSpot are widely recognized for their sensitivity in detecting antigen-specific T cells. A standard setup involves stimulating cells with a peptide of interest. However, advanced setups like cultured and co-cultured ELISpots can take your research to the next level. In this post, we’ll explore these setups and highlight published examples to help inspire your next study.
Cultured ELISpot
What is cultured ELISpot?
Cultured ELISpot involves pre-culturing cells before adding them to the ELISpot plate for the detection of analyte-secreting cells. It’s useful for expanding rare cell populations or allowing certain cellular processes to occur before analyte secretion measurement.
Cultured ELISpot is commonly used to expand an antigen-specific T cell population in vitro before assessing their functionality with ELISpot. PBMCs can be cultured with antigens of interest or other mitogens for a specified time to expand the T cell population. After this incubation, cells are washed and added to an ELISpot plate with the antigen again to measure cytokine secretion.
Chen et al. (2020) utilized this solution in their study on hepatitis B virus (HBV), where they developed a cultured ELISpot assay to detect HBV-specific T cells that were otherwise elusive in chronically infected patients. Using Mabtech’s Human IFN-γ ELISpot kits, the researchers took PBMCs from these patients and compared the traditional ELISpot with the cultured method. In the traditional method, PBMCs were stimulated overnight with HBV peptides in ELISpot plates coated with the capture IFN-γ mAbs and developed as usual. For cultured ELISpot, PBMCs were first stimulated with HBV peptides, IL-7, and IL-2 for 10 days, washed, and then added to the ELISpot plate with HBV peptides before carrying out the assay as usual. They concluded that the cultured ELISpot method had a higher sensitivity for detecting and measuring HBV-specific T cells compared to the traditional ELISpot. This setup highlighted the utility of cultured ELISpot in studying chronic infections where antigen-specific cells can be difficult to detect.
Cells are pre-cultured with specific stimuli for a defined period before being transferred to a FluoroSpot or ELISpot plate, where analyte secretion is then detected.
Read the entire paper and check out another great paper that used cultured ELISpot to answer their research questions:
- Chen et al., Identification of the association between HBcAg-specific T cell and viral control in chronic HBV infection using a cultured ELISPOT assay, Leukoc Biol 2016
- van Pul et al., Breast cancer-induced immune suppression in the sentinel lymph node is effectively countered by CpG-B in conjunction with inhibition of the JAK2/STAT3 pathway, J Immunother Cancer 2020
While cultured ELISpot allows for expanding rare cell populations, co-cultured ELISpot takes a different approach by focusing on the interaction between distinct cell types. Let’s learn more!
Co-cultured ELISpot
What is co-cultured ELISpot?
As the “co-“ prefix suggests, co-cultured ELISpot involves culturing different cell types together to study their interactions and the resulting cytokine secretion. This setup is useful when investigating cellular cross-talk, antigen presentation, as well as immune modulation.
A common co-cultured ELISpot setup is used to study the interactions between T cells and dendritic cells (DCs). DCs are typically loaded with an antigen and co-cultured with T cells to assess T cell activation and cytokine responses.
Uebele et al. (2017) used co-cultured ELISpot and FluoroSpot to explore how different antigen delivery methods to dendritic cells (DCs) influence CD4 and CD8 memory T cell responses. In their study on Staphylococcus aureus, they first isolated monocytes from PBMCs and differentiated them into monocyte-derived dendritic cells (MoDCs). The MoDCs were then either pulsed with staphylococcal protein antigens or transfected with mRNA coding for those antigens. When co-cultured with CD4 and CD8 T cells in IFN‑γ ELISpot or FluoroSpot Plus: Human IFN‑γ/IL-13 plates, these antigen-loaded MoDCs induced cytokine secretion, revealing distinct immune responses based on the delivery method. Pulsing with native proteins promoted a tolerogenic response while transfecting MoDCs with mRNA skewed T cells toward a more inflammatory, Th1-biased response. Their findings highlight the importance of antigen presentation methods in shaping T cell memory responses and the potential applications of co-cultured ELISpot in evaluating immune responses to pathogens like S. aureus.
Co-cultured FluoroSpot involves incubating two or more cell types together to study their interactions and the resulting cytokine secretion.
Read the paper in its entirety and check out other great papers that used cultured ELISpot to provide valuable insights for their studies:
We hope this post has helped inspire how you can take ELISpot and FluoroSpot a step further using cultured and co-culture techniques to gain an even better understanding of the intricacies of immune responses. These methods allow you to either amplify specific immune responses, improving sensitivity, or observe interactions between cell types, providing a more complete understanding of immune mechanisms.
Ready to push your research further? Unlock the full potential of your ELISpot and FluoroSpot assays by integrating cultured and co-cultured setups.