Immunology JoVE (Open Access) Citable · DOI

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

Name: Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR
Uploaded: 2026-06-17
Duration: 8 min 2 s
Description: Immune cell subtype population frequencies can have a large effect on the efficacy of T cell therapies. Current methods, like flow cytometry, have ...

DOI: 10.3791/60465-v

What you'll learn

✓ Perform bisulfate conversion on DNA samples for methylation analysis
✓ Design and execute qPCR assays targeting epigenetic signatures of CD8+, Treg, and Th17 cells
✓ Validate immune cell identity and purity using DNA methylation as a molecular marker

Protocol

Here we describe a robust method of determining immune cell identity and purity through epigenetic signatures detected using quantitative PCR (qPCR). DNA demethylation at a specific locus serves as a unique identifier for a particular cell type and allows for identification of CD8+, regulatory, or Th17 T cells.

Difficulty

advanced

Total time

~2-3 days per sample batch (includes bisulfate conversion overnight and qPCR run)

Model organism

Mouse

Biosafety

BSL-1

Steps

Understand epigenetic markers for immune cell identification

Review the principle that DNA demethylation at specific loci serves as unique identifiers for CD8+, regulatory, and Th17 T cell populations. This foundation explains why methylation status can determine cell identity and purity.

▶ 00:05

Prepare and isolate immune cell samples

Obtain sorted or enriched immune cell populations and prepare genomic DNA for downstream methylation analysis. Sample preparation ensures sufficient DNA quantity and quality for bisulfate conversion.

▶ 00:41

Convert cytosine to uracil via bisulfate treatment

Treat DNA samples with sodium bisulfate to deaminate unmethylated cytosines to uracils while leaving methylated cytosines unchanged. This chemical conversion creates the basis for qPCR discrimination of methylation status.

▶ 01:41

Perform CD8+ and Treg cell methylation qPCR assays

Design and execute qPCR assays targeting epigenetic loci specific to CD8+ T cells and regulatory T cells using bisulfate-converted DNA. Methylation patterns at these loci serve as molecular identifiers for each cell type.

▶ 02:20

Perform Th17 cell methylation qPCR assay

Execute qPCR targeting the epigenetic signature specific to T helper 17 cells on bisulfate-converted DNA. This assay completes the panel for distinguishing Th17 identity from other T cell subsets.

▶ 05:28

Compare methylation data with flow cytometry results

Analyze representative methylation qPCR data alongside flow cytometry data to validate cell purity and identity assignments. Cross-correlation demonstrates the concordance of epigenetic markers with traditional immunophenotyping.

▶ 06:37

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