Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs

Abstract

ELISpot and flow cytometry are two methods often utilized side-by-side for detecting secreted and intracellular cytokines, respectively. Each application has its own advantages and challenges. ELISpot is more sensitive compared to ELISA and appears to be more consistent in detecting IL-10 production than flow cytometry. ELISpot can be used for detecting the secretion of multiple cytokines but not from the same cells simultaneously, whereas flow cytometry allows for the concurrent detection of multiple intracellular cytokines by the same cells. Flow cytometry is a convenient technique allowing for the detection of many cytokines at the same time in a population of cells. The restimulation cocktails used for cytokine detection in flow cytometry are hard on cells and lead to decreased cell viability. Using a live dead dye allows for the exclusion of dead cells when analyzing data. We illustrated the differences between ELISpot and flow cytometry by stimulating cells with two toll-like receptor (TLR) agonists, LPS or Pam₃CSK₄. Both activators increase production of various cytokines, including IL-10, IL-6, and TNF-alpha. The TLR2 antagonist, MMG-11, was used to inhibit this increased cytokine production. We observed some inhibition of IL-6 and IL-10 from Pam₃CSK₄ stimulation in the presence of MMG-11 by flow cytometry. TNF-α remains largely unchanged as its basal expression is high, but there is some reduction in the presence of MMG-11 for both methods. However, IL-10 was difficult to detect by ELISpot given the low seeding density. Overall, both ELISpot and flow cytometry are good methods for detecting secreted and intracellular cytokines, respectively, and should be used as complimentary assays.