Cell cytotoxicity and viability assay

Submitted by :
Submitted to:
Vishal Sonkar
Dr. Manisha
Maneesh kr.
Sachan
Maurya
(Associate
professor)
Cell viability assays
• Cell viability assays measure the number of live, metabolically
active cells in a culture.
• These assays use various methods to determine the number of
viable cells, such as counting cells using a microscope or flow
cytometer, measuring metabolic activity using colorimetric or
fluorometric assays, or assessing membrane integrity using
dye that selectively stain live or dead cells.
• Cell viability assays provide information on the ability of cells
to survive, grow, and carry out their functions under specific
conditions
Cell Viability Assays:

I. Trypan blue exclusion assay

II. MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
III. XTT assay (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide)
IV. Resazurin assay (7-hydroxy-3H-phenoxazin-3-one 10-oxide)
V. Live/dead staining assay ATP assay (adenosine triphosphate)
Trypan blue exclusion assay
• The Trypan blue exclusion assay is a widely used method for
assessing cell viability and quantifying the number of dead cells in a
sample. It is based on the principle that viable cells have intact cell
membranes that exclude certain dyes such as Trypan blue, whereas
dead cells have compromised cell membranes that allow the dye to
penetrate and stain the cells.
• By quantifying the number of dead cells in the sample, the Trypan
blue exclusion assay provides an indirect measure of cell toxicity or
cell death induced by drugs, chemicals, or other substances.
• The Trypan blue exclusion assay is a simple, inexpensive, and
reliable method for assessing cell viability, and is widely used in cell
culture experiments, drug screening, and other fields.
 The Steps involved in the Trypan blue exclusion assay is as follows:

 Harvest cells: Collect cells from culture, blood, or tissue samples and
transfer them to a sterile container.
 Prepare the Trypan blue solution: Prepare a solution of 0.4% Trypan blue
in phosphate-buffered saline (PBS) or another appropriate buffer. The
solution can be filtered through a 0.2-μm filter to remove any particulate
matter.
 Mix the cells with Trypan blue: Mix equal volumes of the cell suspension
and Trypan blue solution. Pipette up and down to ensure a homogeneous
mixture.
 Load the hemocytometer: Place a small volume of the Trypan blue-cell
suspension onto a hemocytometer or a counting slide. Allow the cells to
settle for a few minutes.
 Count the cells: Using a microscope, count the number of live (unstained)
and dead (stained) cells in a defined area of the hemocytometer.
 Calculate cell viability: Calculate the percentage of viable cells as the
number of unstained cells divided by the total number of cells counted.
 Advantages of the Trypan blue exclusion assay Include:

 Simple and cost-effective: The assay is relatively simple and does not require expensive equipment
or reagents.
 Rapid and reliable: The assay can provide results within a few minutes and is widely used to assess
cell viability in a variety of cell types.
 Quantitative: The assay allows for quantitative measurements of cell viability by counting the
number of viable and nonviable cells.
 Versatile: The assay can be used for a wide range of cell types, including bacteria, yeast, and
mammalian cells.
 Compatibility with downstream assays: The assay does not interfere with subsequent assays, such as
cell culture or biochemical assays.

Limitations to the Trypan blue exclusion assay:

 Limited accuracy: The assay relies on manual counting, which can introduce variability and errors
in the results.
 Poor discrimination between necrosis and apoptosis: The assay cannot differentiate between cells
that have undergone necrosis or apoptosis, which can limit its usefulness in some applications.
 Inability to detect early stages of cell death: The assay may not detect early stages of cell death,
such as changes in membrane permeability, before cells become nonviable.
 Requirement for viable cells: The assay cannot be used to assess the viability of cells that do not
take up the dye, such as certain types of bacteria.
 Potential toxicity of Trypan blue: Although Trypan blue is generally considered safe for use in cell
culture, it may have toxic effects on some cell types, particularly when used at high concentrations
MTT assay
The MTT assay measures the reduction of yellow tetrazolium salt to
purple formazan by mitochondrial dehydrogenases in viable cells.
The amount of formazan produced is proportional to the number of
viable cells present in the sample, and a decrease in formazan
production indicates cell death or damage.

Source: https://icbg.ucdavis.edu/ap3/therapeutics-screening-methods
 Steps involved in the process:

 Seed cells: Seed cells in a 96-well plate or another appropriate format at a density
that allows them to reach confluence within the desired experimental time frame.
 Add MTT reagent: Add MTT reagent to each well, such that it is diluted in the
cell culture medium at a final concentration of 0.5 mg/mL.
 Incubate the cells: Incubate the cells at 37°C for 1-4 hours, depending on the
experimental setup and the cell type being used. During this time, MTT is
reduced by mitochondrial dehydrogenases in viable cells, forming insoluble
formazan crystals.
 Remove the medium: Remove the medium from each well and wash the cells
gently with PBS or another appropriate buffer to remove any unreacted MTT.
 Add solubilization solution: Add a solubilization solution, such as dimethyl
sulfoxide (DMSO), to each well to dissolve the formazan crystals.
 Incubate and measure absorbance: Incubate the plate at room temperature for 10-
30 minutes to ensure complete solubilization of the formazan crystals, then
measure the absorbance at 570 nm using a microplate reader.
 Data analysis: Calculate the percentage of cell viability or proliferation by
comparing the absorbance of treated cells to that of untreated control cells.
 Advantages:
 Quantitative: The MTT assay provides a quantitative measure of the number of viable cells
present in a culture.
 Reliable: The MTT assay is widely used and has been shown to be reliable and consistent in
many different types of cells.
 Versatile: The MTT assay can be used with a wide range of cell types and experimental
conditions, making it a versatile tool for cell viability assessment.
 High-throughput: The MTT assay is compatible with high-throughput screening, allowing
for the rapid screening of many different compounds or experimental conditions.
 Cost-effective: The MTT assay is relatively inexpensive compared to other methods for
measuring cell viability.

Limitations:
 Indirect measure of cell viability: The MTT assay measures mitochondrial activity rather
than direct cell viability, which can lead to misinterpretation of the results in some cases.
 Insensitivity to early stages of cell death: The MTT assay may not be able to detect early
stages of cell death, such as changes in membrane permeability, before cells become
nonviable.
 Potential interference: Certain compounds or dyes can interfere with the MTT assay, leading
to inaccurate results.
 Limited information on cellular mechanisms: The MTT assay provides information on the
number of viable cells but does not provide information on the mechanisms underlying cell
death or proliferation.
 Limited dynamic range: The MTT assay has a limited dynamic range and may not be able to
XTT Assay
 The XTT assay is a colorimetric assay used to assess the metabolic activity
of cells and cell viability. The assay relies on the ability of metabolically
active cells to reduce a tetrazolium salt, XTT (2,3-bis-(2-methoxy-4-nitro-
5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), to a formazan dye that can
be quantified spectrophotometrically.

Source: https://www.researchgate.net/figure/Principle-of-XTT-
Steps involved in the process:

Seed cells: Seed cells in a 96-well plate or another appropriate format at a

density that allows them to reach confluence within the desired
experimental time frame.
Add XTT solution: Add XTT solution to each well, such that it is diluted in
the cell culture medium at a final concentration of 0.3-1 mg/mL.
Optionally, an electron-coupling reagent such as PMS can also be added to
enhance the color development.
Incubate the cells: Incubate the cells at 37°C for 1-4 hours, depending on
the experimental setup and the cell type being used. During this time, XTT
is reduced by mitochondrial dehydrogenases in viable cells, forming water-
soluble formazan products that are released into the medium.
Measure absorbance: Remove the medium from each well and measure the
absorbance of the formazan products at 450-500 nm using a microplate
reader.
Data analysis: Calculate the percentage of cell viability or proliferation by
comparing the absorbance of treated cells to that of untreated control cells.
 Advantages:
 Quantitative measurement: The XTT assay provides a quantitative measure of cell
viability, based on the absorbance of formazan products.
 High sensitivity: The XTT assay is sensitive to changes in cell viability and can detect
relatively small changes in cell numbers.
 Compatibility with high-throughput screening: The XTT assay is compatible with high-
throughput screening, which allows for the rapid screening of many different compounds
or experimental conditions.
 Versatility: The XTT assay can be used with a wide range of cell types and experimental
conditions, making it a versatile tool for cell viability assessment.

Limitations:
 Indirect measure of cell viability: The XTT assay measures mitochondrial activity rather
than direct cell viability, which can lead to misinterpretation of the results in some cases.
 Potential interference: Certain compounds or dyes can interfere with the XTT assay,
leading to inaccurate results.
 Limited dynamic range: The XTT assay has a limited dynamic range and may not be able
to accurately measure viability in cultures with low or high cell densities.
 Incubation period: The XTT assay requires an incubation period of several hours, which
may not be suitable for all experimental setups.
Resazurin assay
 The resazurin assay is a colorimetric assay used to assess cell viability and
metabolic activity. The assay is based on the ability of living cells to reduce
resazurin, a blue, non-fluorescent dye, to resorufin, a pink, fluorescent dye,
through mitochondrial enzymes.
 The resazurin assay is widely used in research, drug discovery, and
toxicology studies because it is simple, rapid, sensitive, and non-toxic to
cells. The assay can be performed in multi-well plates, and the protocol is
straightforward. The cells are seeded in a plate, and resazurin solution is
added to the wells. The cells are then incubated, and after a certain period,
the fluorescence or absorbance of the resorufin product is measured using a
fluorescence or spectrophotometric plate reader.
 One advantage of the XTT assay over other viability assays is that it is non-
toxic and does not interfere with cell growth or metabolism, which makes it
suitable for long-term studies.
 Another advantage is that the assay can be used to assess cell
proliferation as well as viability, as the amount of formazan
produced is proportional to the number of metabolically active
cells.
However, the resazurin assay also has some limitations. For
example, the assay is affected by the number of cells in the
assay, and higher cell densities may reduce the sensitivity of
the assay. Furthermore, some compounds or experimental
conditions may interfere with the reduction of resazurin to
resorufin, leading to false results. Despite these limitations, the
resazurin assay remains a useful and popular tool for assessing
cell viability and metabolic activity.
 The general steps for performing the resazurin assay:
 Prepare the cell suspension: Harvest the cells and prepare a single-cell suspension
in the appropriate culture medium.
 Plate the cells: Seed the cells in the wells of a 96-well plate at the desired cell
density.
 Incubate with the test compound: Incubate the cells with the test compound or
drug at the appropriate concentration for the desired duration.
 Add the resazurin dye: Prepare the resazurin dye solution by dissolving it in sterile
phosphate-buffered saline (PBS) or culture medium. Add the resazurin solution to
the wells containing the cells, using a volume that does not exceed 10% of the
culture medium volume in the well.
 Incubate the plate: Incubate the plate for a suitable period of time, usually between
1 to 4 hours, at 37°C in a humidified CO2 incubator.
 Read the absorbance: After incubation, read the absorbance of the wells at the
appropriate wavelength using a microplate reader.
 Analyze the data: Calculate the percentage of cell viability for each well based on
the absorbance values, using the appropriate control samples (e.g., untreated cells,
cells treated with vehicle only, etc.).
 Advantages of the resazurin assay include:
 High sensitivity: The assay can detect changes in cell viability at low concentrations
of test compounds.
 Versatility: The assay can be used to evaluate the effect of different types of
compounds, including drugs, toxins, and environmental pollutants.
 Speed: The assay can be performed quickly and easily, and can be automated for
high-throughput screening.
 Cost-effectiveness: The assay is relatively inexpensive compared to other cell
viability assays.

Limitations to the resazurin assay:

 Non-specificity: The assay does not distinguish between different modes of cell
death, such as necrosis and apoptosis.
 Variability: The reduction of resazurin to resorufin can be influenced by various
factors, such as cell density, culture conditions, and assay duration, which can
introduce variability in the results.
 Toxicity: The assay requires the addition of a potentially toxic dye to the cells,
which can affect cell viability and interfere with the assay results.
 Cell type-dependent: The reduction rate of resazurin to resorufin can vary among
different cell types and can affect the sensitivity of the assay.
Cell cytotoxicity assays
• Cell toxicity assays measure the extent to which a
compound or experimental condition causes damage or
impairs the function of cells.
• These assays can be used to assess different modes of cell
death, such as necrosis, apoptosis, or autophagy, as well as
the effects of compounds on specific cellular processes,
such as DNA synthesis or protein synthesis.
• Cell toxicity assays can also provide information on the
potency and mechanism of action of compounds, as well
as the potential for adverse effects on cells or organisms.
The Cytotoxicity Assays

I. LDH assay (lactate dehydrogenase)

II. MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium)
III. DNA fragmentation assay
IV. Caspase activity assay
V. Micronucleus assay
LDH assay
• The LDH assay is a commonly used assay that measures the
activity of lactate dehydrogenase (LDH) in the culture medium of
cells. LDH is a cytoplasmic enzyme that is released into the
culture medium when cells are damaged or undergo lysis.
Therefore, the amount of LDH released into the medium is a
measure of cell death or damage.
• By measuring the LDH activity in the culture medium, the
number of dead or damaged cells in the sample can be
determined. This information can then be used to assess the
cytotoxic effects of drugs, chemicals, or other substances on cells.
• The LDH assay is a simple, sensitive, and reliable method for
evaluating cell death or damage, and is widely used in drug
discovery, toxicology, and other fields.
 The steps involved in LDH assay is as follows:
 Prepare the cell suspension: Harvest the cells and prepare a single-cell suspension in the
appropriate culture medium.
 Plate the cells: Seed the cells in the wells of a 96-well plate at the desired cell density.
 Incubate with the test compound: Incubate the cells with the test compound or drug at
the appropriate concentration for the desired duration.
 Prepare the LDH assay solution: Prepare the LDH assay solution by diluting the LDH
detection reagent in the assay buffer provided in the kit.
 Collect the supernatant: After the desired incubation period, collect the culture
supernatant from the wells using a pipette, being careful not to disturb the cell
monolayer.
 Add the LDH assay solution: Add the LDH assay solution to the collected supernatant in
each well, and mix gently by pipetting.
 Incubate the plate: Incubate the plate for a suitable period of time, usually between 10 to
30 minutes, at room temperature or 37°C.
 Stop the reaction: Stop the reaction by adding the stop solution provided in the kit.
 Read the absorbance: After stopping the reaction, read the absorbance of the wells at the
appropriate wavelength using a microplate reader.
 Analyze the data: Calculate the percentage of LDH release for each well based on the
absorbance values, using the appropriate control samples (e.g., untreated cells,
maximum LDH release control, etc.).
 Advantages of the LDH assay include:
 High sensitivity: The assay can detect small changes in cell membrane integrity, which
can occur in response to different types of cell damage or death.
 Wide applicability: The assay can be used with a variety of cell types and is not limited to
any specific type of cell.
 Speed: The assay can be performed quickly and easily, and can be automated for high-
throughput screening.
 Cost-effectiveness: The assay is relatively inexpensive compared to other cell viability
assays.

Limitations to the LDH assay:

 Non-specificity: The assay does not distinguish between different modes of cell death,
such as necrosis and apoptosis.
 Interference: The assay can be affected by substances in the culture medium or other
compounds that can interfere with the detection of LDH, which can affect the accuracy of
the results.
 Cell type-dependent: The LDH release rate can vary among different cell types and can
affect the sensitivity of the assay.
 Reproducibility: The assay can be affected by factors such as cell density, culture
conditions, and the duration of incubation, which can introduce variability in the results.
 Live/dead staining
 Live/dead staining is a fluorescence-based assay that utilizes two fluorescent dyes to
distinguish between live and dead cells in a cell population. The assay is based on the
principle that live cells have intact plasma membranes that exclude certain dyes, while
dead cells have compromised plasma membranes that allow dyes to enter the cell.
 The live/dead staining protocol typically involves the use of two dyes: a cell-
permeable esterase substrate, such as calcein AM or green fluorescent protein (GFP),
and a cell-impermeable nucleic acid dye, such as propidium iodide (PI) or ethidium
homodimer-1 (EthD-1). The esterase substrate is cleaved by intracellular esterases in
live cells to produce a green fluorescent signal, while the nucleic acid dye enters dead
cells and binds to DNA to produce a red fluorescent signal.
 The stained cells are then visualized under a fluorescence microscope or flow
cytometer, where live cells appear green and dead cells appear red. The percentage of
live cells in the sample can be calculated based on the ratio of green to red
fluorescence.
 It is important to note that the specific live/dead staining protocol may vary depending
on the cell type, culture conditions, and experimental design. Optimization of staining
conditions is critical to ensure accurate and reproducible results.
 The steps involved in this process:

 Prepare the cell suspension: Harvest the cells and prepare a single-cell suspension in the
appropriate culture medium.
 Seed the cells: Seed the cells in the wells of a 96-well plate or onto a slide or cover slip.
 Prepare the staining solution: Prepare the staining solution by diluting the live/dead staining
dyes in the culture medium or buffer according to the manufacturer's instructions.
 Add the staining solution: Add the staining solution to the cells, using a volume that does
not exceed 10% of the culture medium volume in the well.
 Incubate the cells: Incubate the cells with the staining solution for the appropriate period of
time, usually between 5 to 30 minutes, at room temperature or 37°C in a humidified CO2
incubator.
 Rinse the cells: Rinse the cells with the appropriate buffer to remove any excess dye.
 Mount the cells: Mount the cells on a glass slide or coverslip with an anti-fade mounting
medium.
 Observe the cells: Observe the cells under a fluorescence microscope, using appropriate
filters to visualize the green and red fluorescence.
 Analyze the data: Count the number of live and dead cells based on their fluorescence
intensity and calculate the percentage of live cells in the population.
ATP assay
The ATP assay is a commonly used method for measuring the amount of ATP
(adenosine triphosphate) in a sample. ATP is a molecule that stores and transfers
energy in cells, and is therefore an important indicator of cellular metabolism
and viability.

The basic principle of the ATP assay is as follows:

 Cells are lysed or disrupted to release ATP into the sample.
 The sample is mixed with a luciferin-luciferase reagent, which generates light in the
presence of ATP.
 The amount of light emitted is measured using a luminometer or a microplate reader,
and is proportional to the amount of ATP in the sample.
 By measuring the amount of ATP in the sample, the ATP assay provides an indirect
measure of cell viability and metabolic activity. Changes in ATP levels can indicate
changes in cell proliferation, energy metabolism, or cellular stress.
 The ATP assay is a sensitive, rapid, and non-destructive method for measuring cell
viability and metabolic activity, and is widely used in drug discovery, toxicology, and
other fields. It can also be used to measure the activity of enzymes that utilize ATP, such
as kinases and ATPases.
 Flow cytometry
Flow cytometry is a technique used to analyze and sort cells based on their
physical and chemical characteristics. It is widely used in biological and
medical research to study various aspects of cellular behavior, including
cell size, shape, complexity, DNA content, protein expression, and cell
viability.

The basic principle of flow cytometry is as follows:

 Cells are suspended in a buffer or medium and introduced into a flow
cytometer, a specialized instrument that uses lasers and detectors to
measure various properties of the cells as they pass through a narrow
channel.
 The cells are focused into a single stream using hydrodynamic forces and
passed through the laser beams.
 The lasers illuminate the cells, and the detectors measure the scattered light
and fluorescence emitted by the cells.
 The scattered light and fluorescence signals are converted into electrical
signals, which are processed by a computer to generate a multidimensional
data set of the cells.
 The data can be analyzed using various software tools to identify different
subpopulations of cells based on their physical and chemical characteristics.
 Flow cytometry can be used to analyze large numbers of cells rapidly and
accurately, and can be coupled with cell sorting techniques to isolate
specific subpopulations of cells for further analysis or culture. It is widely
used in immunology, hematology, oncology, microbiology, and other fields,
and has numerous applications in research, clinical diagnosis, and drug
development.
Caspase activity assays
 Caspases are a family of cysteine proteases that play a critical role in
programmed cell death or apoptosis. The activation of caspases leads to the
cleavage of various cellular substrates, resulting in the dismantling of the
cell. The measurement of caspase activity is an important tool for studying
the process of apoptosis and its regulation.
 Caspase activity assays are based on the ability of caspases to cleave
specific peptide substrates that are conjugated to a chromogenic or
fluorogenic group. When the substrate is cleaved by the caspase, the
chromogenic or fluorogenic group is released, resulting in a measurable
signal that can be quantified using a spectrophotometer or fluorometer.
 There are several types of caspase activity assays available, For example,
the caspase-3/7 activity assay uses a substrate containing the tetrapeptide
sequence DEVD and can be used to measure the activity of caspase-3 and
caspase-7, which are key effector caspases involved in apoptosis. The
caspase-8 activity assay uses a substrate containing the sequence IETD to
measure the activity of caspase-8, which is involved in the extrinsic
pathway of apoptosis.
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