Immunology JoVE (Open Access) Citable · DOI

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Name: Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts
Uploaded: 2026-06-17
Duration: 8 min 54 s
Description: Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

DOI: 10.3791/57134-v

What you'll learn

✓ Extract and isolate potato tuber protoplasts via plasmolysis and enzymatic digestion
✓ Prepare nuclear suspensions from protoplasts for flow cytometric analysis
✓ Measure endoreduplication levels in potato tuber cells using flow cytometry
✓ Interpret flow cytometric data to assess DNA ploidy distribution

Protocol

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Difficulty

intermediate

Total time

~3 days (Day 1: plasmolysis ~1 hr; Days 2–3: protoplast generation, harvest, wash, and flow cytometry ~6–8 hrs total)

Model organism

Solanum tuberosum (potato)

Biosafety

BSL-1

Steps

Plasmolyze tuber tissue on Day 1

Incubate potato tuber tissue in plasmolysis solution to induce cell wall separation from the cell membrane, reducing cell debris in subsequent protoplast extraction.

▶ 00:36

Generate and harvest potato protoplasts

Over Days 2–3, enzymatically digest plasmolyzed tuber tissue to release protoplasts, then harvest and wash protoplasts to remove enzymatic debris and contaminants.

▶ 01:40

Prepare nuclear suspensions for flow cytometry

On Day 3, lyse isolated protoplasts and prepare nuclear suspensions with appropriate buffers and staining dyes to enable flow cytometric detection of DNA content.

▶ 03:42

Perform flow cytometric analysis of nuclei

Run prepared nuclear samples through flow cytometry to measure DNA ploidy levels and quantify endoreduplication within potato tuber cells.

▶ 05:07

Interpret endoreduplication results

Analyze flow cytometric histograms to determine the frequency and extent of endoreduplication (polyploidy) in the isolated tuber protoplasts.

▶ 06:31

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