JoVE Logo

Sign In

Abstract

Immunology and Infection

Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR

Published: February 19th, 2020

DOI:

10.3791/60465

1Cell Biology, Life Sciences Solutions, Thermo Fisher Scientific, 2California Polytechnic State University (Cal Poly), 3Epiontis GmbH, Precision for Medicine

Abstract

Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have specific sample requirements, high sample input, are low throughput, and are difficult to standardize, all of which are detrimental to characterization of cell therapy products during their development and manufacturing.

The assays described herein accurately identify and quantify immune cell types in a heterogeneous mixture of cells using isolated genomic DNA (gDNA). DNA methylation patterns are revealed through bisulfite conversion, a process in which unmethylated cytosines are converted to uracils. Unmethylated DNA regions are detected through qPCR amplification using primers targeting converted areas. One unique locus per assay is measured and serves as an accurate identifier for a specific cell type. The assays are robust and identify CD8+, regulatory, and Th17 T cells in a high throughput manner. These optimized assays can potentially be used for in-process and product release testing for cell therapy process.

Explore More Videos

Keywords Immune Cell Identity

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved