---
title: "Freeze/thawing cells"
slug: "freeze-thaw-cells-cut-and-tag"
description: "How to prepare and use frozen, cryopreserved, freeze-thawed cells for CUT&RUN."
tags: ["cut and tag", "cut and run"]
updated: 2026-02-27T15:19:38Z
published: 2026-02-27T15:19:38Z
canonical: "support.epicypher.com/freeze-thaw-cells-cut-and-tag"
---

> ## 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.

# Freeze/thawing cells

> [!NOTE]
> This article also applies to meCUT&RUN and Multiomic CUT&RUN.
> 
> Important Notes
> 
> Use conditions that minimize lysis, which can contribute to elevated background. Ensure [Digitonin is optimized](/v1/docs/optimizing-cell-permeabilization-with-digitonin) for cell types.
> 
> This protocol is for cells only. For nuclei, see “Nuclei Cryopreservation and Thawing Protocol” in [this article](https://support.epicypher.com/docs/nuclei-extraction-protocol-for-cutana-assays). Note that if you intend to use nuclei, we recommend isolating nuclei before freezing rather than freezing cells and trying to isolate nuclei after thawing.

## **Freezing cells**

1. Count cells and [confirm viability, integrity, and morphology](/v1/docs/confirm-high-quality-sample-prep-cut-and-run) using [Trypan Blue staining](/v1/docs/trypan-blue-staining). Spin cells 600 x g, 3 min, room temperature (RT).
2. Remove supernatant. Resuspend in cell culture media with 10% DMSO and aliquot as desired. EpiCypher typically aliquots 5 million cells for 8 reactions, which allows for ~20% sample loss during freeze/thaw.
3. Slowly freeze aliquots (-1˚C per minute) in an isopropanol-filled chiller in a -80°C freezer (e.g. “Mr. Frosty”).

## **Thawing cells**

1. When ready to perform a CUTANATM assay, remove tubes from -80˚C and quickly place on a 37°C block to thaw. Work quickly to avoid cell lysis.
2. When cells are almost thawed, remove from 37˚C and pipette to fully thaw cells.
3. Spin cells at 600 x g, 3 min, RT. Pipette to remove supernatant.
4. Resuspend cells in 105 μL per reaction RT Wash Buffer. Take a 10 μL aliquot to count using [Trypan Blue staining](/v1/docs/trypan-blue-staining). Note that viability may be decreased; focus instead on cell integrity, lysis levels, and total cell counts. If significant sample loss has occurred, spin cells again and resuspend in a smaller volume of Wash Buffer.
5. Continue to ConA bead binding section of the appropriate protocol for your assay.