Validation of Th17 Cell Differentiation from Peripheral Blood CD4+T Cells Through Assessment of mRNA Expression and Cytokine Secretion Using Microplate Reading and Cellular Imaging - "Article Preview" 

CD4+ T cells signal and regulate an immune response to pathogens after interacting with an antigen-MHC (major histocompatibility) complex. Based on several factors, these cells differentiate into distinct phenotypic and functional effector cells, known as T helper cells [Th] – one of which is Th17 cells. These cells are well known for their association with multiple inflammatory and autoimmune disorders. The Th17 cells produce pro-inflammatory cytokine, IL-17 and targeting its pathway reduces the severity of autoimmune diseases. Therefore, there is a growing interest in using Th-17 cells as therapeutic target. Moreover, the Th-17 cells can be both anti-tumor and pro-tumorigenic depending upon the type of cancer. For cancer research, the impact of Th-17 cells needs to be studied and currently it is difficult to produce these cells in good numbers for such studies.  

The current process for isolating Th-17 cells is labor and time-intensive. In this paper, a validated robust method to differentiate peripheral blood CD4+ T cells into functional Th17 cells using a bead-based activation technology is demonstrated. A novel cell imaging multi-mode reader is used to image phenotypic differences between cells exposed to the antibody/cytokine cocktail and negative control cells as differentiation proceeds. Creation of Th-17 cells is confirmed by assessing IL-17F mRNA levels using a fluorescence RNA in situ hybridization (RNA FiSH) assay.  This combination provides a comprehensive solution for the creation and validation of this important class of helper CD4+ T cells.

hIL-17 secretion determination graph

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