---
title: "Troubleshooting nuclei extraction in cell lines"
slug: "troubleshooting-nuclei-extraction"
tags: ["cut and run", "cut and tag"]
updated: 2026-09-02T02:00:07Z
published: 2026-09-02T02:00:07Z
canonical: "support.epicypher.com/troubleshooting-nuclei-extraction"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://support.epicypher.com/llms.txt
> Use this file to discover all available pages before exploring further.

# Troubleshooting nuclei extraction in cell lines

> This article applies to CUT&RUN, meCUT&RUN, Multiomic CUT&RUN, and CUT&Tag.

When it comes to nuclei extraction, not all cell lines are the same. Below, we discuss how nuclei extraction can vary with cell type, and offer suggestions for common issues during and after extraction.

## Which Cell Types Are Easier to Extract Nuclei From?

In general, nuclei extraction is most straightforward for:

- Suspension or semi-adherent cell lines
- Cells with limited cell-cell interactions
- Cells with greater surface area exposed to nuclei extraction buffer

## Which Cell Types Are More Difficult?

The following sample types are often more challenging and may be prone to clumping:

- Adherent cell lines, especially fibroblasts or epithelial cells
- Invasive cancer cell lines
- Cells that grow in 3D structures
- Samples with extensive cell-cell interactions

## General Recommendations for Difficult Samples

If you observe incomplete nuclear extraction (as in **Figure 1**):

- Increase the incubation time in Nuclei Extraction Buffer (for how to prepare our Nuclei Extraction Buffer ([EpiCypher 21-1026](https://epicypher.com/product/cutana-nuclei-extraction-buffer/)) , see [this article](/v1/docs/nuclei-extraction-protocol-for-cutana-assays)).
- Vortex the sample in Nuclei Extraction Buffer for 5 seconds.
- Perform a second incubation in fresh Nuclei Extraction Buffer (see nuclei extraction protocol linked above).
- Apply additional mechanical disruption (e.g., Dounce homogenization or MiltenyiTM gentleMACS tissue dissociation).
- Add 0.5% BSA or 5 mM EDTA to the nuclei extraction buffer to reduce clumping.

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/image-1786415918267.png)

**Figure 1.** MCF7 breast cancer cell line at different stages of nuclei isolation and stained with either acridine orange/propidium iodide (AOPI) or trypan blue. Black arrows indicate cells; red arrows indicate partially extracted nuclei; yellow arrows indicate isolated nuclei.

## My Nuclei Are Different Sizes After Extraction. Did I Do Something Wrong?

Not necessarily. There are many different reasons for heterogeneity in nuclei size, and many of them are independent from your extraction method:

- There are several biological reasons that your nuclei may be different sizes. For example, cancer cells can have increased chromosomal numbers—or even whole genome duplications—affecting the size of the nuclei. Senescence and replication stress can also impact the size and shape of the nuclei.
- Long exposure to hypotonic solutions such as trypan blue, and gradient centrifugation buffers for removing debris, can lead to apparent differences due to nuclei swelling. This does not necessarily indicate poor sample quality, however, as spending too long in hypotonic solutions (like Nuclei Extraction Buffer) can also cause nuclear lysis.

## **To avoid all of these issues:**

After extraction, use the nuclei immediately or [cryopreserve](/v1/docs/nuclei-extraction-protocol-for-cutana-assays#nuclei-cryopreservation-and-thawing-protocol) them. If you are going straight from nuclei extraction to a CUTANATM assay, move the nuclei into PBS or Wash Buffer I/Wash Buffer as soon as possible. If you are cryopreserving, the nuclei can remain in Nuclei Extraction Buffer for slow freezing.

## **References**

Firestone TM, et al. *High-efficiency genomic mapping of chromatin-associated targets with CUT&RUN*. bioRxiv (2024). doi:10.1101/2024.12.03.626419v