# APPLICATION NOTE

# Real-time monitoring of NK cell activity: A novel approach using Celloger® Pro and optimized staining reagents

## Introduction

**Natural Killer (NK) cells** are a crucial part of the innate immune
system, recognized for their inherent ability to identify and eliminate
malignant or virus-infected cells without prior sensitization.1
Evaluating the efficacy of **NK cell-based therapies** is essential in
preclinical studies, where **NK cell killing assays** play a central
role.

These assays measure **NK cell cytotoxicity** by co-culturing NK cells
with fluorescently labeled target cells, with the degree of target cell
lysis serving as an indicator of NK cell killing activity. **Flow
cytometry** or **fluorescence microscopy** is commonly used to quantify
these results, allowing for a precise evaluation of NK cell function,
which is vital for optimizing and validating NK cell products.

Recent advancements in NK cell therapy, particularly with **Chimeric
Antigen Receptor NK (CAR-NK) cells** engineered to better target and
destroy tumor cells, have driven a growing need for more accurate assays
in cancer treatment.2,5 However, the accuracy of NK cell killing assays
is often compromised by technical challenges due to the rapid leakage of
**Calcein-AM**, a commonly used dye for staining target cells.

**Calcein-AM** is widely recognized for its ability to fluorescently
label live cells with minimal impact on their function, making it a
standard tool in various analytical methods including microscopy and
flow cytometry. However, significant leakage of Calcein-AM has been
reported in certain cell types, raising concerns about its
reliability.6,7

In our experiments, similar issues were observed when Calcein-AM was
used to stain target cells during NK cell killing assays. The leakage of
Calcein-AM reduces the overall cell count, regardless of NK cell
activity, making it challenging to accurately assess NK cell-mediated
target cell death.

To address this issue, **CellTracker™ Green CMFDA Dye** was used, a
green fluorescent dye with less leakage and enhanced intracellular
retention compared to Calcein-AM.8 This allowed for more accurate
monitoring and analysis of NK cell-mediated cytotoxicity.

In this application note, we present a method to analyze NK cell
cytotoxicity in real time with improved accuracy and efficiency. This
method uses the automated live cell imaging system **Celloger® Pro**
along with optimized staining reagents.

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## Materials and Methods

In this study, **U-2OS cells** were used as target cells and prepared
under three different conditions:

1.  Stably expressing **EGFP-tagged H2B**
2.  Stained with **2 μM Calcein-AM** (Invitrogen, C1430)
3.  Stained with **3 μM CellTracker™ Green CMFDA Dye** (Invitrogen,
    C2925)

For cells requiring dye staining:

1.  Cells were incubated at **37°C for 30 minutes** in a working
    solution of the staining reagents in serum-free media.
2.  Cells were washed after incubation.

U-2OS cells stably expressing EGFP-tagged H2B did not require additional
fluorescent dye staining.

After preparation:

1.  Target cells were seeded in **48-well plates** at a density of **3 ×
    10⁴ cells per well**.
2.  Cells were co-cultured with **NK-92 cells** (effector cells) at
    varying **effector-to-target (E:T) ratios**:
    -   5:1\
    -   10:1\
    -   20:1

Cell viability was assessed by adding **4 μM Ethidium homodimer
(EthD-1)** (Sigma, 46043-1MG-F).

Images were captured at **1-hour intervals over a 24-hour period** using
a **Celloger® Pro** with a **4X objective lens**.

Experimental results were analyzed using **Celloger® analysis
software**, focusing on overall green and red fluorescence intensities
to evaluate NK cell cytotoxicity.

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## Results

**NK cell cytotoxicity** was analyzed using **NK-92 cells** as effector
cells and **U-2OS cells** as target cells.

To simulate cancer cell metastasis, U-2OS cells -- naturally adherent
cells with solid tumor characteristics -- were detached and suspended
before co-culture with NK-92 cells. Using suspended U-2OS cells to study
NK cell-mediated cytotoxicity, we aimed to gain insights into the
mechanisms by which NK cells target and destroy metastatic cancer cells.

First, we performed an NK cell killing assay with U-2OS cells stably
expressing **GFP:H2B** bound to DNA. Real-time observation of NK-92
cell-mediated cytotoxicity against U-2OS cells was conducted using
**Celloger® Pro** with a **4X lens** over **24 hours**.

As the effector-to-target (E:T) cell ratio increased:

-   Red fluorescence intensity increased (indicating cell death).
-   Green fluorescence intensity decreased.
-   The size of clusters formed by NK-92 and U-2OS cells increased.

\[Image placeholder: Figure 1. NK-92 killing assay with U-2OS cells
expressing GFP:H2B.\]

To quantify NK cell-mediated target cell death:

1.  Red and green fluorescence intensities were measured using
    **Celloger® analysis software**.
2.  The **red-to-green fluorescence ratio** was calculated.

Results confirmed that target cell death increased proportionally with
the **E:T cell ratio**.

Using stable cell lines like U-2OS cells expressing GFP provides highly
accurate results for long-term monitoring but has several drawbacks:

-   Time-consuming generation process\
-   Requires genetic modification\
-   Multiple rounds of selection\
-   Resource-intensive\
-   Limited practicality for high-throughput experiments

Due to these limitations, an alternative approach using live cell
staining dyes was explored.

### Comparison of Calcein-AM and CellTracker CMFDA

Experiments were conducted using two staining reagents:

-   **Calcein-AM**
-   **CellTracker™ Green CMFDA Dye**

As NK cell ratios increased, similar trends were observed:

-   Increased red fluorescence intensity\
-   Decreased green fluorescence intensity\
-   Formation of larger cell clusters

\[Image placeholder: Figure 2. NK-92 killing assay with U-2OS cells
stained with live cell staining dyes.\]

However, the fluorescence intensity ratio in **Calcein-AM-stained U-2OS
cells** increased disproportionately, particularly at an **E:T ratio of
10:1**.

This was due to:

-   Rapid leakage of Calcein-AM\
-   Reduction in live-labeled cell numbers over time\
-   Overestimation of cell death rate

\[Image placeholder: Figure 3. Comparison of dye retention between
CellTracker CMFDA and Calcein-AM.\]

In contrast, **CellTracker CMFDA** demonstrated:

-   Superior intracellular retention\
-   Green fluorescence persistence for over **12 hours**\
-   Significantly reduced leakage compared to Calcein-AM\
-   Reduced artificial elevation of red-to-green fluorescence ratio

This stability enabled more precise long-term tracking of NK cell
cytotoxicity.

------------------------------------------------------------------------

## Conclusion

This study explored an alternative approach to traditional **NK
cell-mediated cytotoxicity assays**, which historically relied on:

-   **Radioactive chromium (51Cr) labeling**
-   **LDH-based assays**
-   **MTT-based colorimetric/enzymatic assays**

While these conventional methods are well-established, they are limited
by:

-   Single time-point measurement\
-   Risks associated with radioactive materials

To address these limitations:

1.  Target cells were labeled with fluorescent dyes.
2.  **Celloger® Pro** was used for real-time monitoring.
3.  Cytotoxicity was quantified using the **red-to-green fluorescence
    intensity ratio**.

Our findings demonstrated that **Celloger® Pro** offers:

-   Real-time monitoring capabilities\
-   Direct observation of cellular changes\
-   Individual cell-level morphological tracking\
-   Enhanced accuracy in cytotoxicity studies

Specifically:

-   **CellTracker CMFDA** showed higher intracellular retention than
    Calcein-AM.
-   It minimized inaccuracies caused by spontaneous dye leakage.
-   It provided greater flexibility compared to generating stable
    GFP-expressing cell lines.

However, NK cell killing assay results may vary depending on the type of
target cells used. Therefore, users must optimize:

-   Selection of staining dye\
-   Dye concentration

to ensure accurate and reliable results.

------------------------------------------------------------------------

## References

1.  Lizeth G. Meza Guzman., "Natural Killer Cells: Tumor Surveillance
    and Signaling", Cancers, (2020):12(4):952.\
2.  Feixue Wang, Jennie Ka Ching Lau., "The role of natural killer cell
    in gastrointestinal cancer: killer or helper" Oncogene volume 40
    (2021):717-730.\
3.  Tayler J. Croom-Perez., "Chapter 5- Kinetic, imaging-based assay to
    measure NK cell cytotoxicity against adherent cells" Methods in Cell
    Biology volume 178 (2023):63-91.\
4.  Öner ÖZDEMIR., "Cell-Mediated Cytotoxicity Assays" Asthma Allergy
    Immunol volume 17 (2019):61-69.\
5.  Isabel Prager, Carsten Watzl., "Mechanisms of natural killer
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    (2019):1318-1329.\
6.  Carlos L. Aparicio, Louis H. Strong, Martin L. Yarmush & Francois
    Berthiaume, "Correction for Label Leakage in Fluorimetric Assays of
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7.  Nelita Elliott, Tae Lee., "Proliferation behavior of E.coli in a
    three-dimensional in vitro tumor model." Integr.Biol volume 3
    (2011):696-705.\
8.  Christopher DiCesare, Israel Biran., "Individual cell migration
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