Cell-mediated cytotoxicity is a cornerstone of the adaptive immune system, allowing our bodies to effectively identify, target, and lyse cells to help contain pathogens. Read this review of the types of cell-mediated cytotoxicity and appropriate measurement methods. See more at https://astartebio.com/

1. Strategies for Accurately
Measuring Cell‑Mediated
Cytotoxicity
A Review of the Types of Cell-Mediated
Cytotoxicity and Appropriate Measurement
Methods
WHITE PAPER
Getting on with Discovery

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Introduction
Cell-mediated cytotoxicity is a cornerstone of the adaptive immune system, allowing our
bodies to effectively identify, target, and lyse cells to help contain pathogens.
Scientists’ability to harness this targeted killing power, first described in reports in the 1960s, is the
reason the immune system has been studied extensively to fight bacteria, viruses, and infections —
including cancer.
T cells and natural killer (NK) cells have evolved to possess the unique ability to circulate as effector cells,
target cells infected by viruses or other intracellular parasites, and kill infected cells — all while causing
minimal damage to surrounding tissue.
Because cell-mediated cytotoxicity is so critical to enhancing the immune system and developing
targeted immunotherapies, scientists need to be able to measure this function accurately in vitro.
There are multiple forms of cell-mediated cytotoxicity and several methods for measuring it, which we
will discuss below.
3 Forms of Cell-Mediated Cytotoxicity
1) Antigen-Specific T Cell-Mediated
Cytotoxicity
Perhaps the most common form of cell-
mediated cytotoxicity used in cancer
immunotherapies, this is employed by drugs
targeting immune checkpoints, like Keytruda®
and Opdivo®.
In this mechanism, antigen-specific T cell
receptors expressing the CD8 glycoprotein bind
tightly to Major Histocompatibility Complex
(MHC) class I molecules expressed by cancer
cells. This binding triggers the CD8+ T cell to
release perforin and granzymes, initiating lysis of
the cancer cells.
T cell receptors expressing CD4 glycoproteins
become helper T cells, which signal the presence
of cancer cells to CD8+ T cells, which can, in turn,
perform cell lysis.
CD8+ T cell
Infected cell
(target cell)
Class I MHC
molecule
CD8
Perforin
Granzymes
TCR
Figure 1. A CD8+ T cell attaching to a target cell to
initiate cell-mediated cytotoxicity.1

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2) Antibody-Dependent Cellular
Cytotoxicity
ADCC relies upon effector cells — typically NK
cells — to bind to antibodies on the surface of
target cells and initiate cell lysis. While NK cell-
mediated ADCC is the most common example,
macrophages, neutrophils, and eosinophils have
also been shown to mediate ADCC.
This mechanism starts with antibodies that are
bound to the surface of an infected target cell.
The Fc receptors on NK cells recognize and bind
to the antibody, initiating the release of cytotoxic
granzymes and ultimately cell lysis.
Certain antibody-based drugs, such as Herceptin®
and Rituxan®, utilize the ADCC mechanism.
NK cell
Fc receptors
Antibodies
Figure 2. Fc receptors on the NK cell recognize and bind
to antibodies on the surface of the target cell.2
3) Natural Killer Cell-Mediated
Cytotoxicity
Unlike the previously described antibody-
dependent adaptive immunity and antigen-specific
cytotoxicity mechanisms, this cell killing method
of the innate immune system requires neither
antibody expression nor antigen expression.
NK cells can directly recognize tumor cells by
employing a variety of receptors to detect cell
alterations caused by infections and stress. Either
a lack of signal through inhibitory receptors or too
much signal from other receptors can initiate the
cytotoxic process through the natural release of
cytokines.
Most important and relevant to fighting cancer are
the surface MHC molecules, which play a critical
role both in controlling the NK cell response and
maintaining NK cell responsiveness for secondary
responses to known pathogens. Loss of surface
MHC molecules leads to a lack of inhibitory signal,
which can result in NK cell killing.
This mechanism is less commonly used in
immunotherapy development. Chimeric antigen
receptors (CARs) are being engineered into NK cells
to take advantage of their cytotoxic capabilities and
serve as an alternative to T cells.
Inflammatory
cytokines
Activating
receptor
NK cell
Figure 3. A variety of NK cell receptors detect target cell
alterations and can trigger the release of cytokines.3

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Chromium-51 (51
Cr) Release
Considered the gold standard since its creation
in 1968, this method is not used as often
today due to the required hazardous materials
handling. In a Chromium-51 release assay, target
cells are labeled with 51
Cr. When introduced to
the effector cells, the target cells release 51
Cr
through cell lysis. The amount of 51
Cr in the
supernatant of the centrifuged sample can then
be measured.
Pros
DDSensitive — the release of 51
Cr is easy to
detect
DDMeasures death of target cells, not death
of killer cells
Cons
UU Some leakage of the label, higher
background
UU Requires hazardous materials
Materials Needed
1. Target cells
2. Effector cells: NK cells, PBMC containing
Nk cells, T cells, macrophages, neutrophils,
or eosinophils
3. Chromium-51
4. Culture medium
5. Buffer (optional)
6. Detergent
7. 96-well U-bottom microplate
8. Gamma counter or liquid scintillation
counter
Lactate Dehydrogenase (LDH)
Release
The LDH release assay measures the amount
of soluble cytosolic enzyme (LDH) released
during cell death using a colorimetric readout.
LDH is present in most living cells, making it a
convenient and reliable marker of cell death.
LDH release reduces NAD+ to NADH and H+ and
oxidizes in the cell culture. The diaphorase then
reduces tetrazolium salt to a red formazan. The
amount of red color measured is representative
of the amount of damaged or dead cells in the
culture.
There is also a fluorometric version of the LDH
release assay that is equally simple and effective.
Pros
DDLDH is more stable than other enzymes
DDNo label required
Cons
UU Release of LDH is not limited to the target
cells
UU High background
Materials Needed
1. Target cells
2. Effector cells: NK cells, PBMC containing
NK cells, T cells, macrophages, neutrophils,
or eosinophils
3. Culture medium
4. Substrate mix
5. Buffer (optional)
6. 96-well flat-bottom microplate
7. Microplate reader
Methods for Measuring Cell‑Mediated Cytotoxicity

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Calcein Release
The calcein release assay involves labeling
target cells with a non-toxic, non-fluorescent
compound, Calcein AM (acetoxymethyl).
Calcein AM can easily penetrate live cells where
it then produces Calcein, a highly fluorescent
compound.
Upon introduction to effector cells, the live cells
that are damaged or killed release Calcein into
the culture. The fluorescence intensity in the
culture can easily and quickly be measured using
a microplate reader.
Pros
DDCan be detected using fluorescent plate
reader
DDLabel is specific to target cells
Cons
UU High background
Materials Needed
1. Target cells
2. Effector cells: NK cells, PBMC containing NK
cells, T cells, macrophages, neutrophils, or
eosinophils
3. Calcein AM
4. Culture medium
5. Buffer (optional)
6. 96-well flat-bottom microplate
7. Microplate reader
Staining to Detect Degranulation
Another popular technique is staining of CD107a
as a means of detecting degranulation of
cytotoxic T cells or NK cells. CD107a is found on
the granules in CTL and NK cells, which contain
granzyme and perforin, two components
involved in the lysis of target cells by these two
effector cells.
Expression of CD107a is transient, but adding
directly labeled anti-CD107a to T cells or NK
cells prior to exposure to target cells allows for
detection of this antigen during degranulation.
You can also add other cell surface markers
to identify subpopulations of cells that are
degranulating.
Pros
DDOnly requires one label
DDLow background
Cons
UU May miss degranulation
UU Flow cytometer needed
Materials Needed
1. Target cells
2. Effector cells: NK cells, PBMC containing NK
cells, T cells, macrophages, neutrophils, or
eosinophils
3. Primary antibody
4. Paraformaldehyde
5. Methanol
6. Buffer (optional)
7. Detergent
8. 96-well U-bottom microplate
9. Fluorescence microscope
10. Flow cytometer

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Flow Cytometry
For a more advanced and real-time method of
detecting cell-mediated cytotoxicity, consider
flow cytometry-based assays. To use flow
cytometry, first label the target cells with a
fluorescent viability dye to differentiate them
from the effector cells.
Pros
DDDeeper analysis
DDHigh sensitivity
Cons
UU Not as easily read in larger assays
Figure 4. K562, a cell line that is readily killed by NK cells, was labeled with CFSE and combined with increasing numbers of
NK cells. Following overnight incubation, the dead cells were labelled using 7-amino-actinomycin D (7-AAD). Flow cytomet-
ric analysis was used to determine the percentage of dead cells (7-AAD-stained) among the target cells (CFSE-stained) while
excluding the NK cells.
Materials Needed
1. Target cells
2. Effector cells: NK cells, PBMC containing NK
cells, T cells, macrophages, neutrophils, or
eosinophils
3. Primary antibody
4. Culture medium
5. 96-well U-bottom microplate
6. Centrifuge
7. Flow cytometer

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Conclusion
The method you choose for measuring
cell‑mediated cytotoxicity should factor in your
research goals, target cells used, equipment
availability, and measurement and analysis
expertise. While some methods can easily
generate data from 96 wells (luminescent
assays using a plate reader), other methods are
better suited to a smaller number of samples
that provide more extensive data that may
provide needed insights.
Always account for spontaneous death or
release of any label used. Calculation of the
percent cytotoxicity uses spontaneous release
to set a baseline. Samples that are killed by a
toxic compound or detergent are used to set
the maximum. The classic calculation is:
Be sure to run your experiments in triplicate
to control for experimental errors and bias.
Studies that aim to maximize this important
T cell function may lead to improved therapies.
Image Sources
1
Adapted from original illustration by Dananguyen via Creative Commons
2
Adapted from original illustration by Satchmo2000 via Creative Commons
3
Adapted from Kristy C. Newman & Eleanor M. Riley (April 2007). Whatever turns you on: accessory-cell-dependent activation of
NK cells by pathogens. Nature Reviews Immunology 7, 279-291. Accessed from www.nexcelom.com
signal from test sample – signal from spontaneous
----------------------------------------------------------------------------------------- x 100
signal from max lysis – signal from spontaneous

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