# Analysis of Nocodazole-induced Cytotoxicity using **Celloger® Mini Plus**

## Introduction

**Cytotoxicity** refers to the degree of damage to cells caused by chemical substances or physical factors. Measuring it through a **cytotoxicity assay** is essential for drug development and biological research. Cells undergo complex signaling pathways that causes various cell death processes such as **apoptosis**, **necrosis**, and **necroptosis**. However, most cytotoxicity assays are measured at the end-point that makes it difficult to study the dynamic response of cells to drugs.

In this application note, we aimed to examine the performance of a cytotoxicity assay using **real-time imaging**. Cells treated with various concentrations of **Nocodazole**, the anti-cancer drug, were stained with fluorescent dye during cell death, then monitored with **Celloger® Mini Plus**. It was observed through time-lapse imaging that apoptosis increased in a Nocodazole dose-dependent manner, and the degree of apoptosis was quantitatively measured and graphed using the **Analysis software** provided with the **Celloger® Mini Plus**.

## Method

**HeLa cells** were counted using **Facscope B (Curiosis Inc.)**, an automatic cell counter, and seeded at **1 × 10⁴ cells/well** in a **48 well plate**. After culturing overnight, cells were treated with various concentrations (**16.625 nM**, **31.25 nM**, **62.5 nM**, **125 nM**, **250 nM**) of **Nocodazole**.

At this point, **CellTox green dye (Promega, G8742)**, which binds to DNA of cells with impaired membrane integrity during cell death, was added to the sample. Using **Celloger® Mini Plus** installed inside an incubator, cell images were acquired every **1 hour for 48 hours**, then the images were analyzed using the **Analysis software**.

The **cell death rate (%)** was calculated as:

- **Fluorescence coverage** (= dead cell) ÷ **bright field confluency** (= total cell)

## Result

As a result of treating the cells with **nocodazole** by different concentrations and imaging them over time, the morphology of cells changed in a concentration-dependent manner and the confluency shrinkage was observed in **bright field imaging** (Figure 1A, B). In addition, fluorescence imaging of dead cells stained by **CellTox dye**, measuring its coverage and quantifying the cell death showed that the proportion of fluorescent cells increases with apoptosis (Figure 1C).

[Image placeholder: Figure 1. Analysis of Nocodazole dose-dependent cell death using time-lapse imaging.]

### Figure 1 Descriptions

- **(A)** Merged bright field and fluorescence images for each concentration of **Nocodazole**. The images are shown at **24-hour intervals**, and green fluorescence indicates the dead cells. *(Scale bar: 200 um)*  
- **(B)** The **confluency (%)** graph of total cell over time.  
- **(C)** The graph of **cell death rate (%)** over time.

## Conclusion

**Celloger® Mini Plus** is a live cell imaging device that can simultaneously perform **bright field** and **fluorescence imaging** in an incubator. The system has a fully motorized camera that enables imaging of various positions at a set interval programmed by the user. The **confluency graph over time** can be acquired by calculating the confluency of captured images using the **Analysis software**. Furthermore, by measuring the **fluorescence coverage**, the degree of apoptosis according to the concentration of **Nocodazole** can be quantified.

**FOR RESEARCH USE ONLY** and not for use in diagnostic procedures.  
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