RESAZURIN IN VITRO CELL VIABILITY ASSAY

Animal cell and tissue culture assignment

Submitted by: Syed Muhammad Zaki Haider (BSBT-025-F18)

Submitted to: Dr. Moazzam Ali (Assistant professor)

Institute of Biochemistry and Biotechnology (IBB)

University of the Punjab (PU), Lahore.
 RESAZURIN IN VITRO CELL VIABILITY ASSAY 01/10/2021

C
ell viability refers to the amount of healthy cells present in any sample(Kamiloglu et al., 2020).
These very assays that determine cell viability most often are also used to determine the cell
proliferation in a cell population as well(Stoddart, 2011). Good viability levels account for good
proliferation levels, which are positive indicators of optimum cellular growth(Aslantürk, 2017).
Determination of cell viability is very important in cell and tissue culture. Determination of cell
viability may be used to screen the cellular response against a toxin, drug or chemical agent under
consideration, used in toxicology and pharmacology in cytotoxicity experiments, check the
effect of a therapeutic agent that targets cells like that of cancer and inhibition of cancer cell growth in cancer
research, it may act as the main purpose of toxicity assay based experiments and alternatively, can delineate the
correlation between the cell behavior and cell growth(Stoddart, 2011). In vitro cell viability assays are cheap,
animals are not necessarily needed for them, are inexpensive, swift and have potential for automation as well
(Aslantürk, 2017).

CLASSIFICATION OF CELL VIABILITY ASSAYS:

Possible factors that influence the selection of an appropriate cell viability assay include cost, sensitivity, speed,
the required equipment and type of cells under consideration (Shokrzade & Kordi, 2017). Safety, quickness,
reliablity, efficiency, and zero interference with the compound being tested are some top of the list feature of an
ideal cell viability assay(Aslantürk, 2017). Some major types of cell viability assays are mentioned below:
I. Dye exclusion assays (Kamiloglu et al., 2020)
• Trypan blue(Kamiloglu et al., 2020)
• Eosin(Kamiloglu et al., 2020)
• Congo red(Kamiloglu et al., 2020)
• Erythrosine B(Kamiloglu et al., 2020)
II. Colorimetric assays(Kamiloglu et al., 2020)
• MTT assay(Kamiloglu et al., 2020)
• MTS assay(Kamiloglu et al., 2020)
• XTT assay(Kamiloglu et al., 2020)
• WST-1 assay(Kamiloglu et al., 2020)
• WST-8 assay(Kamiloglu et al., 2020)
• LDH assay(Kamiloglu et al., 2020)
• SRB assay(Kamiloglu et al., 2020)
• NRU assay(Kamiloglu et al., 2020)
• CVS assay(Kamiloglu et al., 2020)
III. Fluorometric assays(Kamiloglu et al., 2020)
• Alamar Blue (Resazurin) assay(Kamiloglu et al., 2020)
• CFDA-AM assay(Kamiloglu et al., 2020)
IV. Luminometric assays(Kamiloglu et al., 2020)
• ATP assay(Kamiloglu et al., 2020)
• Real-time viability assay(Kamiloglu et al., 2020)

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 RESAZURIN IN VITRO CELL VIABILITY ASSAY 01/10/2021

These methods are categorized so on the base of the end result indication (i.e color, luminescence, fluorescence,
binding of dye). Based on different functions taking place inviable cells like working of enzymes, permeability
of cell membrane, cell adherence, ATP synthesis, co-enzyme production, and nucleotide uptake (Kamiloglu et
al., 2020; Thangaraj, 2016). Each one of these methods have their own significance, history, pros and cons. We
will dive deep into the Resazurin fluorometric cell viability assay.

FLUOROMETRIC CELL VIABILITY ASSAY:

Fluorometric assays were developed in 1990s, works on the principle of “enzymatic conversion of a
nonfluorescent compound to a fluorescent one by viable cells”(Kamiloglu et al., 2020). This conversion is
followed by fluorescence, this inceptive signal is measured to find out the quantity of viable cells. Fluorometric
assays are preferred over colorimetric and the traditional dye exclusion assays as they offer higher sensitivity,
higher ease of use, lesser expense as they are cheaper and are applicable to both adherent and suspended cells.
Some possible disadvantages of this technique are the fluorescent interference by applied test compounds and it
does not give reliable results with newly thawed cells. Fluorometric assay may use a fluorescence microscopy,
fluorometery, microplate reader or flow cytometery in order to check cell viability by fluorescence. Now let’s
have a look at the resazurin or alamar blue fluorometric assay.

ALAMAR BLUE IN VITRO CELL VIABILITY ASSAY:

The core principle of alamar blue assay is comprising of non-specific, enzymatic reduction of resazurin
(nonfluorescent blue colored dye) into the resorufin (pink colored fluorescent compound) by viable cells,
with the aid of mitochondrial enzymes or other enzymes like diaphorases or esterases(O’Brien et al., 2000).
Resazurin was first discovered by Weselsky and in late 1920s and was then used to measure bacterial
infestation in milk, being an indicator of cellular metabolism. As Viable cells convert resazurin to resorufin,
there is an increase in fluorescence and the color of medium changes due to extraction of the reduced product
from living cells into the medium. Measurement sensitivity can range from 50 to 50,000 cells, using 530–570
nm as excitation wavelength and 580– 620 nm as emission wavelength (Kamiloglu et al., 2020). Thus, more
fluorescence means more resorufin in cells, and more cell viability. After done with experiment, cells can be
returned to the culture medium because of nontoxicity of resazurin reagent to cells (Kamiloglu et al., 2020;
V. Kuete et al., 2017).

SYED MUHAMMAD ZAKI HAIDER (BSBT-025-F18) 2

 RESAZURIN IN VITRO CELL VIABILITY ASSAY 01/10/2021

In vitro Toxicology Assay Kit from Sigma–Aldrich(Kamiloglu et al., 2020), AlamarBlue kit by Bio-
Rad(Kamiloglu et al., 2020), AlamarBlue Cell Viability Reagent made by Thermo Fisher
Scientific(Kamiloglu et al., 2020) etc. are some commercial kits that are manufactured by these companies for
carrying out cell proliferation and viability assays (Kamiloglu et al., 2020).

PREPARATIONS:
Alamar blue reagent can be prepared by dissolving Alamar Blue powder dissolved phosphate buffer saline
(PBS at pH 7.4) to 4mg/ml (Kamiloglu et al., 2020). Storage temperatures comprise of either 4◦C for short-term
storage and -20◦C for long-term storage (Kamiloglu et al., 2020; Markossian et al., 2004). Culture
medium/Resazurin solution can be made by taking 90µl pre incubated cells+medium and 10µl resazurin (total
volume for each well in a 96 well microplate becomes 100µl) (Gilbert & Friedrich, 2017). Filter sterilization can
also be done by passing the solution through a 0.2µm filter (Gilbert & Friedrich, 2017). Make sure to protect the
solution from light.

APPARATUS:
Ultrapure water, Cultured cell line, Multichannel pipette, Tissue culture-compatible 96 well plates,
fluorometer, Fluorescent reader with excitation of 530-560nm and emission of 590nm, 100% reduced form of
alamar Blue Reagent as a positive control by autoclaving alamar blue reagent in cell culture medium , in a ratio
of 1 volume of alamar Blue Reagent for 10 volumes of mammalian cell culture media for 15 minutes, Negative
control (only culture media) for background signal check (Thermo Fisher Scientific).

PROTOCOL:
Starting off with the cells, after splitting them in the wells of the plate containing the culture medium, cells are
incubated at least for 5 hr at 37◦C and 5% CO2 (Kamiloglu et al., 2020). This will make them reach their log phase
of growth. Make sure to add negative controls to check background fluorescence and positive control as well
(containing 100% reduced resazurin). After the incubation is done, 10%v/v alamar blue is added to each well
(Kamiloglu et al., 2020). As for adherent cells, the old media can be removed and the 100µl mastermix solution
can be added, containing 90µl media and 10µl alamar blue(Gilbert & Friedrich, 2017). Once alamar blue is
added to the wells, make sure to cover the plate with aluminum foil for light protection (Kamiloglu et al., 2020).
The plate is incubated again at 37°C, 5% CO2 for 4 hrs. during this time, viable cells will reduce resazurin to
resorufin. After incubation time is done, remove the plate and shake it for 10s to uniformly distribute resorufin
within the well(Gilbert & Friedrich, 2017). Then determine the fluorescence with excitation wavelength set at
530-560nm and emission wavelength set at 590nm (Thermo Fisher Scientific ).

CALCULATION AND OBSERVATION:

Pink color of medium in the wells will indicate the reduction of resazurin by viable cells. Plotting the
fluorescence at 590nm against cell number will give us cell concentration calibration. Growth curve can be
created by calculating cell density from previously done calibration.
Percentage viability can be determined by following formula:
% Viability = Mean ODsample/Mean ODblank × 100. (OD for optical density)
% cell growth = Mean ODsample/Mean ODblank × 100.
% growth inhibition= 100 - %cell growth

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