This presentation summarizes the cell viability assays and their application in a precise manner

1. Seminar Presentation On:
Principles And Applications of
Cell Viability Assays
Presented By:
Harsha Negi
M.Pharm (Pharmacology) Scholar, 1st Semester
SPER, Jamia Hamdard 29/12/2021

2. Contents
◦ Definition 1
◦ Classification 2
◦ Dye Exclusion Assay 3-10
◦ Colorimetric Assay 11-22
◦ Fluorometric Assay 23-28
◦ Luminometric Assay 29-39
◦ Flow Cytometry Assay 40-44
◦ Summary 45-47
◦ References 48

3. Definition
• Cell viability is defined as the number of healthy cells in a sample
• The measurement of cell viability plays an important role for all forms of cell culture
• Cell viability assays are essentially used for screening the response of the cells against a drug or a
chemical agent
• Pharmaceutical industry widely uses viability assays to evaluate the influence of developed agents on
the cells
• 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:
o enzyme activity
o cell membrane permeability
o cell adherence
o adenosine triphosphate (ATP) production
o co-enzyme production
o nucleotide uptake activity
1

4. Classifications
Dye
Exclusion
Assays
Luminometric
Assays
Flow
Cytometric
Assays
Colorimetric
Assays
Fluorometric
Assays
2

5. DYE EXCLUSION ASSAYS
3

6. Introduction
• 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
• It is based on the principle that viable cells have an intact cell membrane which can exclude the
dye whereas the dead cells take up the dye as their membrane is no longer able to control the
passage of macromolecules
• For these assays, although staining procedure is quite straightforward, experimental procedure
may be time-consuming in case of large sample sizes
4

7. Trypan Blue Stain Assay
• Trypan blue stain assay was initially developed in 1975 to measure viable cell count
• The stain is a large negatively charged molecule
• It is one of the simplest assays that are used to determine the number of viable cells in a cell
suspension
• Principle:
• 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
5

8. • Reagent preparation:
◦ 0.4% trypan blue stain + phosphate-buffered saline (PBS) or serum-free medium are
obtained
◦ Trypan blue stain should be stored in dark and filtered after prolonged storage
◦ Trypan blue stain binds to serum proteins and cause misleading results, serum-free medium
should be used to obtain reliable results
• Protocol:
Cell suspension to be tested is centrifuged for 5 min
Supernatant is discarded and the pellet is resuspended in 1-ml PBS
solution or serum free medium
6

9. 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
Following incubation, a drop of the mixture is applied to a
hemocytometer, which is placed on the stage of a
binocular microscope
Viable (unstained) and nonviable (stained) cells in the
hemocytometer are counted separately
7

10. • 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
• The percentage of viable cells is calculated using the following equation:
% Viable cells =
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠 𝑝𝑒𝑟 𝑚𝑖𝑙𝑙𝑖𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑎𝑙𝑖𝑞𝑢𝑜𝑡
𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑝𝑒𝑟 𝑚𝑖𝑙𝑙𝑖𝑙𝑖𝑡𝑒𝑟 𝑜𝑓 𝑎𝑙𝑖𝑞𝑢𝑜𝑡
× 100
8

11. Fig 1: Different steps involved in the Trypan blue assay
9

12. Fig 2: Live/Dead cells under the influence of trypan
blue
10

13. COLORIMETRIC ASSAYS
11

14. Introduction
• 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
• Most widely applied colorimetric assays include:
• MTT: 3-[4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
• MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
• XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
• WST-1: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt
• WST-8: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt
• LDH: lactate dehydrogenase
• SRB: sulforhodamine B
• NRU neutral red uptake
• CVS crystal violet stain
12

15. MTT Assay
• MTT assay is simple colorimetric test of cell proliferation and survival. It was developed by
Mosmann (1983) and adapted by Cole (1986) for measuring chemosensitivity 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 the first homogeneous cell viability assay that was designed for 96-well plates for high-
throughput screening
• In MTT assay, the tetrazolium salt is reduced to insoluble formazan dye by dehydrogenase
enzyme present in the viable cells at 37°C
• 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
• A variety of methods have been used to solubilize the formazan product, stabilize the color,
avoid evaporation, and reduce interference by phenol red and other culture medium
components
13

16. • 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
• The dead cells lose the ability to reduce tetrazolium salts into colored formazan products
• 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
Fig 3: Reduction of MTT to formazan crystals
14

17. • Reagent Preparation:
• MTT is dissolved in Dulbecco’s phosphate buffered saline (DPBS) at pH 7.4 (5 mg/ml)
• The solution is then 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
• Solubilization solution is prepared with 40% (v/v) DMF containing 2% (v/v) glacial acetic acid
under ventilated fume hood
• Sodium dodecyl sulfate (SDS) (16% [w/v]) is added to this solution and pH is adjusted to 4.7
• Solubilization solution should be stored at room temperature in order to prevent precipitation of
SDS and in case of precipitation it can be heated to 37 °C for resolubilization
15

18. • 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 crystal are dissolved in 100 µl of solubilization
solution and the absorbance is measured at 570 nm with a multiplate reader
16

19. Fig 4: MTT Assay
17

20. • Calculation:
• The percentage of cell viability is calculated using the following equation
% Viability =
Mean ODsample
Mean ODblank
× 100
18

21. SRB Assay
• Sulforhodamine B (SRB) assay was developed in 1990 to evaluate the cytotoxicity of anticancer
drugs
• SRB is a bright-pink aminoxanthene dye with two sulfonic groups that bind to amino-acid
residues under mild acidic conditions, and dissociate under basic conditions
• This protocol is based on the binding ability of SRB to cellular proteins, which is fixed using TCA
(trichloroacetic acid)
• The protein-bound dye is then dissolved in Tris-base (tris(hydroxymethyl) aminomethane)
solution and the absorbance values measured at 510 nm are used to determine the number of
viable cells
19

22. • Reagent preparation:
• 10% (w/v) TCA solution, 0.057% (w/v) SRB dissolved in 1% (v/v) acetic acid, 1% (v/v) acetic
acid solution and 10 mM unbuffered Tris-base solution at pH 10.5 are prepared for fixation,
staining, washing, and dissolution steps, respectively
• Protocol:
Cell suspensions seeded to 96-well plates (19,000 cells/well) with or without the test
compound (10 µl dissolved in 10% DMSO) are incubated at 37◦C in a humidified
incubator with 5% CO2 for 72 hr
The cells attached to the bottom of the wells are fixed with addition of 100 µl of cold
TCA and subsequent incubation at 4◦C for 1 hr. After 1 hr, the plate is washed four
times with slow-running tap water
20

23. The excess water is removed using paper towel and a blow dryer is used to dry the
plate completely
Afterward, 100 µl of the SRB solution is added to the cells and the plate is incubated
at room temperature for 30 min
After 30 min of incubation, the unbound SRB stain is removed by washing the wells
four times with acetic acid solution. Again, a blow dryer is used to completely dry the
plate and then SRB stained cells are dissolved in 200µl of unbuffered Tris-base
solution
Another option is to incubate the plate for 30 min in Tris-base solution for complete
solubilization of SRB dye. Then, the absorbance values are recorded at 510 nm
using a plate reader
21

24. • Calculation:
• The percentage of cell-growth and growth inhibition are calculated using the following equations:
% Cell growth =
Mean ODsample
Mean ODblank
× 100
% Growth inhibition = 100 − % Cell growth
22

25. FLUOROMETRIC ASSAYS
23

26. Introduction
• Fluorometric assays were developed in 1990s as an alternative to exclusion dyes and
colorimetric methods
• Fluorometric assays including resazurin and 5-carboxyfluorescein diacetate acetoxymethyl
ester (5-CFDA-AM) assays may be performed with a fluorometer, fluorescence microplate
reader, fluorescence microscope, or flow cytometer
• 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 esterases
• 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
• These assays are advantageous over dye exclusion and colorimetric assays as they are
more sensitive
24

27. 5-CFDA-AM Assay
• 5-carboxyfluorescein diacetate acetoxymethyl ester (5-CFDA-AM) is another compound used
in fluorometric cell viability assays
• It is a target of intracellular nonspecific esterase enzymes in living cells
• Following the nonspecific enzymatic activity of esterases, 5-CFDA-AM is converted into
fluorescent substance carboxy-fluorescein, which is polar and nonpermeable through the
cellular membrane of living cells
• Examples of commercially available 5-CFDA-AM probes are as follows:
• 5-CFDA-AM (5 mg) from Invitrogen
• 5-CFDA, AM from Synchem
• 5-CFDA (5-Carboxyfluorescein diacetate, single isomer,100 mg) from Biotium
25

28. Fig 5: Schematic illustration of the principles of 5-carboxyfluorescein diacetate acetoxymethyl ester (5-
CFDA-AM) assay. 5-CFDA-AM is a target of intracellular nonspecific esterase enzymes in living cells.
Following the nonspecific enzymatic activity of esterases, 5-CFDA-AM is converted into fluorescent
substance carboxyfluorescein (CF), which is polar and nonpermeable through the cellular membrane of
living cells. Dead or dying cells lack the esterases and membrane permeability control so that they do
not have any fluorescent signal
26

29. • Reagent preparation:
• 5-CFDA, AM stock solution of 4 mM can be prepared in anhydrous DMSO. Stock solution can
be aliquoted and stored at –20◦C but the solution must be protected from light and moisture
• Protocol:
Culture medium is aspirated from the wells of adherent cells and diluted 5-CFDA,
AM solution, 4 µM working solution, is added to the respective cells
Cells are split into the wells using serum- and amino acid-free culture medium and
then equal amount of 8 µM 5-CFDA, AM working solution is added to the
corresponding wells
For suspension cell cultures, 8 µM 5-CFDA, AM working solution can be prepared
via 1:500 dilution of 4 mM stock solution in serum- and amino acid-free culture
medium
27

30. The percentage of cell growth and growth inhibition can be calculated using the
equations described in the previous sections
If exact cell number calculation is necessary, a cell number standard curve should
be prepared for each cell type for the analysis of obtained values
Cells are incubated with 5-CFDA, AM for 30 min at 18–22◦C in the dark
Following incubation, fluorescence is measured at excitation and emission
wavelengths of 493 and 541 nm, respectively, using a fluorescence plate reader
28

31. LUMINOMETRIC ASSAYS
29

32. Introduction
• In luminometric assays, a persistent and stable glow-type signal is produced following the
addition of reagent
• These methods comprise ATP and real-time viability 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
30

33. ATPAssay
• ATP bioluminescence was 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. When the ATP synthesis is interrupted and
remaining ATP is depleted by ATPases immediately once the cells loose membrane integrity
and cell viability
• For the detection of ATP concentration, luciferase assay can be applied as ATP is a necessary
component for the oxidation reaction of luciferin. ATP-coupled luciferase reaction can be
summarized as follows:
31

34. • In the luminometric ATP cell viability assays, cells first get permeable to ATP so that luciferase
enzyme can interact with intracellular ATP
• Then intracellular ATPases are inactivated and finally the light is measured via luminometers to
determine the intracellular ATP levels
• 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
• Examples of commercially available luminometric ATP assays are as follows:
• ATP Assay Kit – Luminometric from Assay Biotech
• ATP Determination Kit from ThermoScientific
• Luminescent ATP Detection Assay Kit from Abcam
• CellTiter-Glo Luminescent Cell Viability Assay from Promega,
• Rapid Luminometric ATP Assay Kit from AAT Bioquest
32

35. Fig 6: Schematic illustration of the principles of ATP assay
33

36. • Protocol:
Cells are split into 96-well plates in 100 µl (25 µl for 384-well plate) culture medium
containing desired treatment compounds or conditions
After desired incubation or treatment period, plates are equilibrated to room
temperature for 30 min
Equal volume of assay buffer (100 µl for 96-well plate and 25 µl for 384-well plate) is
added to each well and incubated on an orbital shaker at room temperature for
10–20 min
If the kit has separate solutions (like Luminescent ATP Detection Assay Kit from
Abcam),50 µl of detergent–ATPase inhibitor buffer is added first and after incubation
50 µl luciferin–luciferase buffer is added and incubated (96-well plate)
34

37. Following incubation, luminescence is measured by a luminometer at 560 nm
wavelength
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
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
35

38. Real Time Viability Assay
• Real-time viability assay is a new approach of luciferase method and is the only cell viability
method that allows to monitor the cell viability in real time
• In this method, cell-permeable pro-substrate and luciferase are added into the culture medium
as well but cells are not lysed to release the intracellular ATP
• Instead, viable cells uptake pro-substrate and convert it into “substrate” that diffuses into the
culture medium. Then, luciferase enzyme uses diffused substrate and generates luminescent
signal
• This method can be applied for both continuous measurement applications and end point
assays
• Examples of commercial real-time viability assay kits are RealTime Glo MT Cell Viability Assay
from Promega and MSCGlo Real Time from HemoGenix
36

39. • Protocol:
37
Cells are split into the wells with medium containing desired treatment compound
and real-time viability assay buffer
Cells are then incubated at 37◦C and continuous luminescence measurements are
recorded every 30 min or every hour depending on the experimental design
Real-time cell viability assay buffers are stable at 37◦C up to 72 hr but an
optimization step is necessary for each cell type to determine the time period in
which limiting factor pro-substrate is consumed totally

40. 38
For end-point experiments, real-time viability buffer is added into the wells at the
end of the treatment conditions
Cells are incubated with the buffer at 37◦C for 10 min to 1 hr and then luminescent
signal is measure
For cell viability calculation, standard ATP measurement or percentage cell growth
can be used

41. 39
Fig 7: RealTime-Glo MT Cell Viability Assay overview

42. FLOW CYTOMETRY ASSAYS
40

43. Introduction
• The principle of flow cytometry is characterizing or phenotyping the cells within a liquid flow
through lasers
• Flow cytometry allows simultaneous measurement of the changes in cell morphology by
forward and side light scatter, which makes the technology uniquely suited for measuring the
complex progression of cell death
• Flow cytometry is a quantitative single cell analysis in which cells can be characterized
depending on the size, granularity, and whether carrying a specific fluorescent molecule or not
• Major flow cytometric assays include membrane asymmetry (e.g., annexin V and F2N12S
staining assays), membrane permeability (e.g., nucleic acid and inclusion and exclusion dyes),
and mitochondria assays
41

44. Membrane Permeability Assays
• Analyzing membrane integrity is important not only for mammalian cell culture studies to decide
cell viability but also for food production research such as analyzing ethanol stress during wine
production, for fertility labs to monitor the membrane integrity of sperms
• Apoptotic cells keep their membrane integrity and they are resistant to exclusion dye while
necrotic cells can be detected with exclusion dyes
• Membrane permeability assays are usually a combination of two types of dyes: one staining the
dead cells and a second one staining living cells
• Membrane permeability dyes can be categorized as exclusion dyes, inclusion dyes, and
monomeric cyanine nucleic acid stains
42

45. Inclusion Dyes:
• Inclusion dyes require intracellular enzymatic functionality in addition to the membrane integrity
• Cytoplasmic esterases cleave the nonfluorescent molecules and yield fluorescent compounds.
Examples of inclusion dyes are fluorescein diacetate, carboxyfluorescein, and calcein
• Protocol for calcein staining-
o Reagent preparation :
• Calcein powder is dissolved in cell culture grade DMSO to 5 mM
• Small aliquots of dissolved solution can be stored at 20◦C
• Like fluorometric staining reagents, calcein is sensitive to buffers and solutions containing
esterases. It is not recommended to use intracellular fixation protocols together with calcein
dyes
43

46. • Protocol:
Stock calcein solution (5 mM) is diluted to 10 nM final concentration using culture
medium
Cells are resuspended in the diluted calcein solution and incubated for 30 min at
37◦C
Cells are washed twice after incubation and measured with a flow cytometer at
excitation and emission wavelengths of 488 and 520 nm, respectively. Percentage
of the living cells can be calculated with one of the package software, such as
FlowJo
44

47. S.No Assay Principle Advantages Disadvantages Application
1. Dye
exclusion
assays
Determination
of membrane
integrity
Simple ∙ Rapid ∙
Inexpensive ∙ Versatile
∙ Require small number
of cells
Time-consuming and
labor-intensive for large
number of samples ∙
Counting errors may occur
due to poor cell dispersion,
incorrect dilution,
contamination etc∙ Cannot
differentiate between
healthy cells and viable
cells that lost their function
∙ Trypan blue is toxic to
mammalian cells
Cell
enumeration,
screening of
drugs,
cytotoxicity,
chemosensitivity
testing
2. Colorimetric
assays
Determination
of metabolic
activity
Accurate ∙ Rapid
∙ Sensitive ∙ Reliable ∙
Applicable to both cell
suspensions and
adherent cells
MTT may interfere with
cell culture medium, it is
highly toxic to cells ∙
Incubation time, cell type,
and cell count may affect
the measurements in MTS
assay
Screening of
anti-herpes
simplex virus
compounds,
cytotoxicity, cell
proliferation
45
Summary Of The Cell Viability Assays

48. S.No Assay Principle Advantages Disadvantages Application
3. Fluorometric
assays
Nonspecific
cleavage of a
nonfluorescent
compound into
a fluorescent
compound by
cellular
esterases
Simple ∙ Inexpensive ∙
Safe ∙ Rapid ∙
Applicable to a number
of cell types ∙
Applicable to both cell
suspensions and
adherent cells
Possible fluorescent
interference caused by the
applied test compounds ∙
Cannot be applied to
newly thawed cells ∙ Light
sensitive
Study of α-
glucosidase and
in the prenatal
and postnatal
detection of
Pompe's
disease,
cytotoxicity study
4. Luminometric
assays
Correlation
between a
bioluminescent
reaction and
the effect of a
tested
compound
Simple ∙ Fast ∙
Luminescent signal is
quite stable ∙ Signal
can be measured
within a few hours ∙
Very specific ∙ Can be
applied with very few
cells (>50 cells)
Assay is prone to results
with technical errors such
as pipetting errors
Measurement of
enzyme activity,
determination of
cell proliferation
and cytotoxicity
46

49. 47
S.No Assay Principle Advantages Disadvantages Application
5. Flow
cytometric
assays :
Membrane
permeability
assays
Determination
of membrane
integrity and
permeability
• Monomeric cyanine
nucleic acid stains
are very specific
double-stranded
nucleic acid dyes
• Monomeric cyanine
nucleic acid stains
are compatible with
fixation protocols
Apoptotic cells keep their
membrane integrity and
they are resistant to
exclusion dyes ∙ Calcein
(inclusion dyes) is
sensitive to buffers
containing esterases ∙
Calcein staining and
propidium iodide staining
cannot be combined with
fixation protocols ∙ Light
sensitive
Diagnosis and
post treatment
monitoring of
certain diseases
such as B and T
lymphoblastic
leukemia/
lymphoma

50. References:
◦ Helgason CD, Miller CL. Basic cell culture protocols. Totowa, NJ.: Humana Press; 2005
◦ Kamiloglu S, Sari G, Ozdal T, Capanoglu E. Guidelines for cell viability assays. Food Frontiers.
2020 Sep;1(3):332-49
◦ Stoddart MJ. Cell viability assays: introduction. Mammalian cell viability. 2011:1-6
◦ Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L. Cell viability
assays. Assay Guidance Manual. 2016 Jul 1
◦ Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation
and cytotoxicity assays. Journal of immunological methods. 1983 Dec 16;65(1-2):55-63
◦ Takeuchi H, Baba M, Shigeta S. An application of tetrazolium (MTT) colorimetric assay for the
screening of anti-herpes simplex virus compounds. Journal of virological methods. 1991 Jun
1;33(1-2):61-71
◦ Gilbert DF, Friedrich O. Cell viability assays. Springer New York; 2017
48

51. Thank You