---
title: "What advice do you have for counting my cells/nuclei?"
slug: "what-advice-do-you-have-for-counting-my-cells-nuclei"
updated: 2026-09-02T01:55:48Z
published: 2026-09-02T01:55:48Z
canonical: "support.epicypher.com/what-advice-do-you-have-for-counting-my-cells-nuclei"
---

> ## 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.

# What advice do you have for counting my cells/nuclei?

Accurate cell and nuclei counts are critical for CUT&RUN, CUT&Tag, and related assays, but one common challenge is distinguishing cells/nuclei from debris when using trypan blue. (Note: for tissue and/or frozen samples, we recommend using propidium iodide (with acridine orange) rather than trypan blue. See [this](/v1/docs/can-fluorescent-markers-be-used-for-counting-nuclei-for-cutana-assays) article for more information on working with PI.)

**Note:** we strongly recommend counting with an automated cell counter, like the Countess (Thermo Fisher). Counting manually and/or relying on the cell counts produced by a FACS-sorting machine can result in inaccurate estimates.

**How does trypan blue stain present?**

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/Slide1(3).jpeg)

**Figure 1.** AOPI- and trypan blue-stained examples of cells in varied states of nuclei extraction. A) Intact cells prior to nuclei extraction. White halo around cell body = alive, non-permeable cell membrane. Nucleus of cell should not be blue. B) Failed nuclei extraction leaves permeable and dying cells. C) Partial nuclei extraction: cell membrane (arrow) remains. D) Red arrows: complete nuclei extraction. Green arrow: swollen nuclei—should be <20% of sample.

For more on trypan blue staining in general, see [this article.](/v1/docs/trypan-blue-staining)

## **Why do I see excessive debris after nuclei extraction?**

What appears to be debris is often trypan blue precipitation, not cellular material—however, the presence of this precipitation can still make counting difficult. Trypan blue can precipitate under several conditions:

- High salt concentrations (e.g., 10X PBS) **(Figure 2)**.
- Excess spermidine
- Delayed counting after trypan blue addition: even when salt concentrations are low, precipitation may occur if samples are left sitting for approximately 10 minutes after trypan blue is added. EpiCypher’s Nuclei Extraction Buffer ([EpiCypher 21-1026](https://www.epicypher.com/product/cutana-nuclei-extraction-buffer/)) does not typically cause trypan blue precipitation when samples are counted within 10 minutes.

## **Recommendations for Accurate Counting**

- Count samples promptly: after adding trypan blue, count your cells or nuclei as soon as possible. Delays can allow trypan blue precipitates to form, which may lead to inaccurate counts.
- Before use, incubate trypan blue in a 37C water bath/heat block, and then centrifuge for 30 seconds.
- Be sure you are pulling trypan blue from the top of the solution.
- Trypan blue can be syringe-filtered (0.22 um) if there is growth or persistent precipitate after warming.

![](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/base64-converted-image-1781638979373.png)

**Figure 2.** Trypan blue-stained nuclei in 1X PBS (left) are easier to count than trypan blue-stained nuclei in 10X PBS (right), because the higher salt concentration of the 10X PBS causes the trypan blue to precipitate.