1.DYE EXCLUSION ASSAYS: Dye exclusion assays are the simplest methods that are based on utilization of different dyes such as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not by dead cells. For these assays, although staining procedure is quite straightforward, experimental procedure may be time-consuming in case of large sample sizes. a. Trypan blue stain assay: Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is still used as a confirmatory test for measuring changes in viable cell number caused by a drug or toxin. Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used to determine the number of viable cells in a cell suspension. Principle: The principle of this assay is that living cells have intact cell membranes that exclude the trypan blue stain, whereas dead cells do not. Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy to determine whether cells include or exclude the stain. A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm. Reagent preparation: To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline (PBS) or serum-free medium are obtained. Trypan blue stain should be stored in dark and filtered after prolonged storage. As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium should be used to obtain reliable results. Assay Protocol: The cell suspension to be tested is centrifuged at 100 g for 5 min. The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free medium. Then, one portion of this cell suspension is mixed with one portion of trypan blue stain. The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will lead to cell death and hence reduced viability counts. Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on the stage of a binocular microscope. Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these cells are counted separately. . Calculation: After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot is determined by multiplying the total number of viable cells by 2, which is the dilution factor for trypan blue. Similarly, total number of cells per milliliter of aliquot is determined by addition of number of viable and nonviable cells and multiplying it by 2. Then, the percentage of viable cells is calculated using the following equation. % Viable cells = Total number of viable cells per milliliter of aliquot × 100. Total number of cells per milliliter of aliquot 2.COLORIMETRIC ASSAYS: Colorimetric assays

1. Principles of cell viability assays
Submitted to:
Dr. B. M. Gurupadayya.
Professor and Head of the department,
Dept. of Pharmaceutical chemistry.
JSS College of Pharmacy.
Mysuru.
Submitted by:
N. Surendra chowdary
M. Pharm, 1st Year
Pharmaceutical Analysis.
JSS College Of Pharmacy, Mysuru

2. Contents:
. INTRODUCTION,
 DEFINITION ,
 CLASSIFICATION,
i) Dye Exclusion Assays
ii) Colorimetric assays
iii) Fluorometric assays
iv) Luminometric assays
v) Flow cytometric assays
 ADVANTAGES AND DISADVANTAGES OF THE ABOVE ASSAYS,
 REFERENCES.

3. INTRODUCTION:
 Cell-based assays are often used for screening collections of compounds to determine if the test
molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to
cell death.
 Cell-based assays also are widely used for measuring receptor binding and a variety of signal
transduction events that may involve the expression of genetic reporters, trafficking of cellular
components, or monitoring organelle function. Regardless of the type of cell-based assay being
used, it is important to know how many viable cells are remaining at the end of the experiment.
 There are a variety of assay methods that can be used to estimate the number of viable eukaryotic
cells. The methods described include: tetrazolium reduction, resazurin reduction, protease
markers, and ATP detection.

4. Contd….
 All of these assays require a incubation reagent with a population of viable cells to convert a
substrate to a colored or fluorescent product that can be detected with a plate reader. Under most
standard culture conditions, incubation of the substrate with viable cells will result in generating
a signal that is proportional to the number of viable cells present.
 When cells die, they rapidly lose the ability to convert the substrate to product. That difference
provides the basis for many of the commonly used cell viability assays.

5. DEFINITION:
 Cell viability is a measure of the proportion of live, healthy cells within a population. Cell
viability assays are used to determine the overall health of cells, optimize culture or experimental
conditions, and to measure cell survival following treatment with compounds, such as during a
drug screen.
 The measurement of cell viability plays an important role for all forms of cell culture.
Sometimes it is the main purpose of the experiment as in toxicity assays, or it can be used to
correlate cell behaviour to the number of cells.
 Cell viability assays are essentially used for screening the response of the cells against a drug or
a chemical agent. In particular, pharmaceutical industry widely uses viability assays to evaluate
the influence of developed agents on the cells.
 Researchers apply various types of assays in order to screen the outcome of a developed
therapeutics that often target cancer cells.

6. Contd….
 There are several types of assays that can be used to determine the number of viable cells.
 These assays are based on various functions of cells including
 Enzyme activity,
 Cell membrane permeability,
 Cell adherence,
 Adenosine triphosphate (ATP) production,
 Co-enzyme production,
 Nucleotide uptake activity.

7. CLASSIFICATION :
 Cell viability assays may be broadly categorized as following :
(1) Dye exclusion assays
(2) Colorimetric assays
(3) Fluorometric assays
(4) Luminometric assays
(5) Flow cytometric assays.

8. 1.DYE EXCLUSION ASSAYS:
 Dye exclusion assays are the simplest methods that are based on utilization of different dyes such
as trypan blue, eosin, congo red, and erythrosine B, which are excluded by the living cells, but not
by dead cells.
 For these assays, although staining procedure is quite straightforward, experimental procedure
may be time-consuming in case of large sample sizes.
a. Trypan blue stain assay:
 Trypan blue stain assay has initially been developed in 1975 to measure viable cell count and is
still used as a confirmatory test for measuring changes in viable cell number caused by a drug or
toxin.
 Trypan blue stain, a large negatively charged molecule, is one of the simplest assays that are used
to determine the number of viable cells in a cell suspension.

9. Principle:
 The principle of this assay is that living cells have intact cell membranes that exclude the trypan
blue stain, whereas dead cells do not.
 Cell suspension is mixed with the trypan blue stain and examined visually under light microscopy
to determine whether cells include or exclude the stain.
 A viable cell will have a clear cytoplasm, whereas a nonviable cell will have a blue cytoplasm.
Reagent preparation:
 To perform the trypan blue stain assay, 0.4% trypan blue stain and phosphate- buffered saline
(PBS) or serum-free medium are obtained.
 Trypan blue stain should be stored in dark and filtered after prolonged storage.
 As trypan blue stain binds to serum proteins and causing misleading results, serum-free medium
should be used to obtain reliable results.
FIGURE : Determination of cell viability with trypan blue assay.

10. Assay Protocol:
 The cell suspension to be tested is centrifuged at 100 g for 5 min.
 The supernatant is discarded and the pellet is resuspended in 1-ml PBS solution or serum-free
medium.
 Then, one portion of this cell suspension is mixed with one portion of trypan blue stain.
 The mixture is allowed to stay at room temperature for 3 min. It is important to note that the cells
should be counted within 3–5 min of mixing with trypan blue, as longer incubation periods will
lead to cell death and hence reduced viability counts.
 Following the incubation, a drop of the mixture is applied to a hemocytometer, which is placed on
the stage of a binocular microscope.
 Viable cells will remain unstained, and nonviable cells will stain, in the hemocytometer and these
cells are counted separately.

11. .
Ref:
https://researchtweet.com/trypan-blue-exclusion-test-
of-cell-viability/
Fig: protocol for Trypan blue stain assay

12. Calculation:
 After counting viable and nonviable cells, the total number of viable cells per milliliter of aliquot
is determined by multiplying the total number of viable cells by 2, which is the dilution factor for
trypan blue.
 Similarly, total number of cells per milliliter of aliquot is determined by addition of number of
viable and nonviable cells and multiplying it by 2.
 Then, the percentage of viable cells is calculated using the following equation.
% Viable cells = Total number of viable cells per milliliter of aliquot × 100.
Total number of cells per milliliter of aliquot

13. 2.COLORIMETRIC ASSAYS:
 Colorimetric assays are based on the measurement of a biochemical marker to determine the
metabolic activity of the cells.
 In these assays, the colorimetric measurement of cell viability is carried out spectrophotometrically.
 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT),
 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2- (4- sulfophenyl)-2H-tetrazolium
(MTS) assays are among the most widely applied colorimetric assays.
 These assays are simple and economical, and can be applied to both cell suspensions and adherent
cells.
a. MTT ASSAY:
 MTT assay is a simple colorimetric test of cell proliferation and survival, which was developed by
Mosmann (1983) and adapted by Cole (1986) for measuring chemo sensitivity of human lung cancer
cell lines.
 The assay is based on the conversion of MTT into formazan crystals by living cells, which shows
mitochondrial function. It is well known as the first homogeneous cell viability assay was designed
for 96-well plates for high- throughput screening.
 Since then, MTT tetrazolium assay technology has been widely adopted.

14. Principle.
 In MTT assay, the yellow tetrazolium salt is reduced to insoluble purple formazan dye by
dehydrogenase enzyme present in the viable cells at 37◦C.
 Further, the insoluble formazan salt is dissolved by the addition of solubilizing agents, and the
colored product is quantitatively measured at 570 nm using a spectroscopic multiplate reader.
 The dead cells can not reduce tetrazolium salts into colored formazan products.
 Where,Viable cells with active metabolism convert MTT into a purple-colored formazan
product with an absorbance maximum near 570 nm. Thus, the intensity of the colored product is
directly proportional to the number of viable cells present in the culture.
 Various solubilization methods include the use of acidified isopropanol, dimethyl sulfoxide
(DMSO), dimethylformamide (DMF), sodium dodecyl sulfate (SDS), and combinations of
detergent and organic solvent.
fig: Reduction of MTT to formazan crystals

15. Fig: Principle of MTT assay
1. 2.
3.

16. Reagent preparation:
 MTT solution is prepared by dissolving MTT in Dulbecco’s phosphate buffered saline (DPBS) at
pH 7.4 (5 mg/ml).
 This solution is filtered and sterilized through a 0.2-μm filter into a sterile and light-protected
container. MTT solution should be stored at –20◦C until analysis or at 4◦C for immediate use and
should be protected from the light.
Assay Protocol:
 Cell suspensions seeded to 96-well plates (100 μl/well) with or without the test compounds are
incubated at 37◦C in a humidified incubator with 5% CO2 for required exposure time.
 MTT solution of 10 μl is added to each well to reach a final concentration of 0.45 mg/ml and
incubated at 37◦C for 1–4 hr.
 After incubation, the formazan crystals are dissolved in 100 μl of solubilization solution and the
absorbance is measured at 550 nm with a multiplate reader.

18. Fig: Change in cell morphology after exposure to MTT (0.5 mg/ml).
Panel A shows a field of cells photographed immediately after addition of the MTT solution. Panel B
shows the same field of cells photographed after4 hours of exposure to MTT. It shows a change in
cell morphology and the appearance of formazan crystals.
Calculation:
The percentage of cell viability is calculated using the following equation:
% Viability = Mean OD sample
Mean OD blank.
× 100
(OD means optical density)

19. 3.FLUOROMETRIC ASSAYS:
 Fluorometric assays are developed in 1990s as an alternative to exclusion dyes and colorimetric
methods. Fluorometric cell viability methods are based on the nonspecific cleavage of a
nonfluorescent compound such as fluorescein diacetate which fluoresces following its cleavage by
cellular esterase's.
 Nascent fluorescent signal is then measured to determine the amount or the ratio of the viable
cells.
 Fluorometric assays are easy to perform and relatively cheap but fluorescent interference caused
by the applied test compounds is possible.
1. Resazurin (Alamar blue) assay:
 The resazurin-based test was first used to examine the sanitary state of milk in the late 1920s.
After that, it has been used for plant metabolism studies , semen quality evaluations , and
antifungal susceptibility tests . It has also been a valuable method for analyzing toxicants owing to
several advantages of the assay.

20. Principle:
 Alamar blue fluorometric assay is based on the nonspecific, enzymatic, irreversible reduction of
the compound by viable cells. Following the enzymatic reaction within the cells, alamar blue or
resazurin(less fluorescence) is reduced into pink resorufin(high fluorescence), and extracted from
the living cells into the medium.
 The extracted compound results in a change in the colour of the medium and colour change can be
measured from 50 up to 50,000 cells in a linear range, using 530–570 nm for excitation/580– 620
nm for emission fluorescent filters.
Dehydrogenase enzyme

21. Reagent preparation:
 Alamar blue solution can also be prepared from the powder instead of using commercially
available kits. Alamar Blue high-purity powder is dissolved in PBS (pH 7.4) to 0.15 mg/ml.
Alamar blue solution can be sterilized via filtering and can be stored at 4◦C for short-term storage
and at –20◦C for long-term storage.
Assay Protocol:
 Prepare cells and test compounds in opaque-walled 96-well plates containing a final volume of
100 µl/well. An optional set of wells can be prepared with medium only for background
subtraction and instrument gain adjustment.
 Incubate for desired period of exposure.
 Add 20 µl resazurin solution to each well.
 Incubate 1 to 4 hours at 37°C.
 Record fluorescence using a 560 nm excitation / 590 nm emission filter set.

22. Figure: protocol for Resazurin (alamar blue) assay.

23. 4.Luminometric assays:
 The bioluminescence assays are based on the correlation between a bioluminescent reaction and
the effect of a tested compound. This effect can be an increase in cell proliferation or cell death.
 Bioluminescent measurements are performed using luminometers since 1970s. Modern
luminometers carry a photon counter and the obtained signal is proportional, but not equal, to the
emitted photons.
a. ATPAssay:
 ATP bioluminescence has initially been developed to determine whether there was a linear
relationship between cultured cell number and measured luminescence using the luciferin–
luciferase reaction.
 Intracellular ATP is a valid indicator of cell viability.

24. Principle:
 ATP can be used to measure cell viability
since only viable cells can synthesize ATP.
 ATP can be measured using the CellTiter-
Glo® Luminescent Cell Viability Assay with
reagents containing detergent, stabilized
luciferase and luciferin substrate. The
detergent lyses viable cells, releasing ATP
into the medium. In the presence of ATP,
luciferase uses luciferin to generate
luminescence, which can be detected within
10 minutes using a luminometer (Figure 2).
 Luminescent signal is quite stable and can be
measured within a few hours and most of the
assays are very specific that the signal can be
measured even from 50 cells.

25. Commercially available luminometric ATP assays:
 ATP Assay Kit – Luminometric from Assay Biotech,
 ATP Determination Kit from Thermo Scientific,
 Luminescent ATP Detection Assay Kit from Abcam,
 Cell Titer-Glo Luminescent Cell Viability Assay from Promega,
 Rapid Luminometric ATP Assay Kit from AAT Bio quest.
Protocol:
 Prepare opaque-walled multi well plates with mammalian cells in culture medium. Volumes and
cell number should be optimized for experimental conditions.
 If desired, prepare control wells containing medium without cells to determine background
luminescence.
 Add test compound to experimental wells, and incubate according to your culture protocol.
 Equilibrate the plate and its contents to room temperature for approximately 30 minutes.
 Add a volume of CellTiter-Glo® 2.0 Reagent equal to the volume of cell culture medium present
in each well (e.g., for a 96-well plate, add 100µl of CellTiter-Glo® 2.0 Reagent to 100µl of
medium containing cells).
 Mix the contents for 2 minutes on an orbital shaker to induce cell lysis (see Appendix for more
information on mixing).
 Allow the plate to incubate at room temperature for 10 minutes to stabilize the luminescent signal.
 Record luminescence.

26. For calculation,
 ATP standard curve is prepared using the standard ATP stock solution. A standard curve with 10
pM to 10 µM range is anticipated to be sufficient for comparison. Working solution is also added
to the standard curve wells and the standard curve plate is incubated at room temperature for the
same period as the experimental groups.
 Following incubation, luminescence is measured by a luminometer at 560 nm wavelength.
Fig: protocol for CellTiter-
Glo® Luminescent Cell Viability
Assay
Ref: https://www.promega.in/products/cell-health-assays/cell-viability-
and-cytotoxicity-assays/celltiter_glo-2_0-
assay/?catNum=G9241#protocols

27. 5.FLOW CYTOMETRIC ASSAYS:
 Flow cytometry is a quick and reliable method to quantify viable cells.
 Determining cell viability is an important step when evaluating a cells response to drug
treatments or other environmental factors.
 It is also often necessary to distinguish dead cells in a cell suspension in order to exclude them
from analysis.
 Dead cells can generate artifacts as a result of non-specific antibody staining or through uptake
of fluorescent probes.
 One method to test cell viability is using dye exclusion. Live cells have membranes that are still
intact and exclude a variety of dyes that easily penetrate the damaged, permeable membranes of
non-viable cells
a. Propidium Iodide Staining:
 Propidium iodide (PI) staining is a viability dye flow cytometry method used to assess cell
viability.
 PI staining can provide information about the cell cycle and DNA content of the population.

28. Principle:
 Propidium iodide solution is a bacterial fluorescence staining dye and can be applied for
microbial cell viability assay in different principles.
 It is an ethidium bromide analog that emits red fluorescence upon intercalation with
double-stranded DNA. Though PI does not permeate viable cell membranes, it
passes through injured cell membranes and stains the nuclei.
 PI is often used in combination with a fluorescein compound, such as CFDA, for
simultaneous staining of viability and membrane injury.
Fig: Principle involved in Propidium iodide (PI) staining

29. Protocol:
Required Equipment:
 Fluorescence microscope (blue or green excitation light, red emission filter)
 Flow cytometer (488 nm or 533 nm laser, red emission filter)
 Micropipette (20 µL, 1000 µL)
Staining procedure:
 Allow PI solution to stand at room temperature for 30 min for thawing. Solution should be
protected from light).
 Resuspend the organisms with PBS(-) or saline and adjust the number of cells to 106cells/mL(flow
cytometry) or 108-109cells/mL(microscopy).
 Add 1 µL of PI solution into the 1 mL of microbial cell suspension and vortex gently to mix.
 Incubate the microbial cells at room temperature for 5 min.
 Analyze the stained cells by flow cytometer or under a microscope.

32.  Fundamental Techniques in Cell culture Laboratory Handbook 3rd edition – SigmaAdrich.
 Wiley Online Library-Guidelines for Cell Viability Assays- Senem Kamiloglu.
 www.ncbi.nlm.nih.gov.in
 https://pubmed.ncbi.nlm.nih.gov
 www.cdcso.gov.in
 Images- Internet search.
 https://www.rndsystems.com/resources/protocols/flow-cytometry-protocol-analysis-cell-
viability-using-propidium-iodide
 https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/175/383/792
14dat.pdf
 https://www.slideshare.net/VidyaNani/principles-applications-of-cell-viability-assays-mtt-
assays
 https://www.promega.in/products/cell-health-assays/cell-viability-and-cytotoxicity-
assays/celltiter_glo-2_0-assay/?catNum=G9241#protocols
References: