Defining iNKT Cell Subsets and Their Function by Flow Cytometry

Abstract

This article discusses methods to assess invariant natural killer T (iNKT) cell subsets isolated from the thymus, as well as the spleen, the liver, and the lung. iNKT cells can be subdivided in distinct, functional subsets based on the transcription factors they express and the cytokines they produce to regulate the immune response. Basic Protocol 1 focuses on characterizing murine iNKT subsets ex vivo by flow cytometry by evaluating the expression of lineagespecifying transcription factors such as PLZF and RORγt. The Alternate Protocol describes a detailed approach to define subsets based on expression of surface markers. This approach can be very useful for maintaining the subsets alive, without fixing them, in order to isolate them for downstream molecular assays such as DNA/RNA isolation, genome-wide analysis to assess gene expression (such as RNA-seq), assessment of chromatin accessibility (for instance, by ATAC-seq), and assessment of DNA methylation by whole-genome bisulfite sequencing. Basic Protocol 2 describes the functional characterization of iNKT cells, which are activated in vitro with PMA and ionomycin for a short period of time and subsequently stained and characterized for production of cytokines, such as IFNγ and IL-4, by flow cytometry. Basic Protocol 3 describes the process of activating iNKT cells in vivo using α-galactosyl-ceramide, a lipid that can be recognized specifically by iNKT cells, allowing assessment of their functionality in vivo. Cells are then isolated and directly stained for cytokine secretion.

Deciphering the TET3 interactome in primary thymic developing T cells

“…Mice were dissected and thymi were isolated and placed immediately in tubes containing ice-cold T cell medium as previously described. 133 , 134 The tubes were placed on ice until the tissue was processed. Each thymus was placed inside a 70 μm cell strainer (BD Falcon, cat no: 352350) inside a 60 mm plate (BD Falcon) and lysed using the pestle of a 1 mL syringe without needle.…”

“…Each thymus was placed inside a 70 μm cell strainer (BD Falcon, cat no: 352350) inside a 60 mm plate (BD Falcon) and lysed using the pestle of a 1 mL syringe without needle. 133 , 134 Cells were measured in a Novocyte cytometer (Acea Agilent) using the NovoExpress software (Agilent).…”