CHAPTER 1

INTRODUCTION TO IN VITRO TOXICOLOGY AND CELL CULTURE

In vitro toxicity testing is the scientific analysis of the effects of toxic chemical
substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing
methods are employed primarily to identify potentially hazardous chemicals and/or to
confirm the lack of certain toxic properties in the early stages of the development of
potentially useful new substances such as therapeutic drugs, agricultural chemicals, direct
food additives, cosmetics, etc. In vitro models offer a number of ethical and economic
advantages over animal testing. These assays are being used earlier in toxicity testing
pipelines, often to determine risk assessment and/or set up controls that will ultimately spare
animal life. The combination of lower-cost and higher throughput assays can help bring
products to market faster. Additionally, in vitro models give researchers an advantage in
understanding the biological processes involved in a toxic response sooner than if they were
depending on the visual inspection of a live animal. In vitro techniques represent tools that
the toxicologist can utilize at various stages of drug development to design and select
compounds to perform specialized evaluations and to address preclinical or clinical issues
that may arise. The use of in vitro technologies contributes to the scientific quality and
economy of the safety assessment process as well as to the toxicologists' commitment to the
three R’s of reduction, refinement, and replacement, and a fourth R, responsibility.
Advancements in cell culture have given researchers a viable alternative and/or
complement to live animal testing. In vitro investigations in specialized cell cultures have
been used to clarify the mechanism of toxic action of chemicals on a particular target organ.
Cell culture is the technique by which either prokaryotic or eukaryotic cells are grown under
controlled conditions. In practice the term "cell culture" refers to culturing of cells derived
from multicellular eukaryotes, especially animal cells. The cellular systems used in toxicity
studies include primary cells, genetically modified cells, immortalized cells, stem cells, and
cells in different stages of differentiation and transformation, co-cultures of different cell
types, etc. The focus is on the cellular models used to study chemical toxicity, specific
toxicity end points at levels including molecular, and the extrapolation of data obtained from
in vitro models to the context of in vivo. Thus, in vitro toxicology can potentially replace
animal use in toxicological evaluations to a very great extent.

1
 Based on the characteristics of the source of cells, cell cultures are classified as follows:
Primary cell culture: One that is started from tissues/organ freshly removed from the
organism. They are generally prepared by treating the original tissue with cell dispersing
agents and planting the cell suspension on a glass/plastic substrate, where the cells adhere and
grow. When cells from primary culture are serially transferred it causes a selection of a single
cell type to become predominant and multiply at a constant rate. The primary cell culture is
then said to have originated a cell strain. Life span is limited to 50 to 60 passages and then it
dies out and the strain comes to an end. During multiplication of a cell strain some cells
become altered and acquire a different morphology, grow faster and have unlimited life. It is
now designated as a cell line.
Established cell lines or continuous cell culture: eg Hep2, ME-180. One in which
the cell nuclei contain chromosome numbers other than the diploid number (heteroploid). The
cells are capable of indefinite replication, not anchorage dependent, not inhibited by density
of population, malignant, and capable of growing in defined media consisting of low
molecular weight nutrients without protein supplements. The transformed cell lines or
modified cell lines are those cells which are either derived from tumor cells or have been
manipulated in some way i.e., transfection with oncogenes or treatment with carcinogens.

1.1 Types of mammalian cultures

 Explant cultures: Cell culture initiated from a fragment of tissue/ organ planted in the
medium/plasma clot. The cells migrate out and form a monolayer, and the original tissue
becomes necrotic.
 Monolayer cultures: Consists as a layer of cells growing on the surface of a culture vessel.
Limitation here is the surface area available for the cells to adhere.
 Suspension Culture: Anchorage-independent cells grow while suspended in fluid medium.
This would yield very large populations of cells.
 Micro-carrier cell cultures: Used for large scale propagation of anchorage-dependent cells
that cannot be cultivated in suspension, but attached to the surface of carrier particles.

2
 1.2 Advantages of cell culture

Reduction of animal use Cytotoxicity and screening of pharmaceutics, cosmetics,

etc.
Reagent saving Reduced volumes, direct access, lower cost
Physico-chemical environment Controlled pH, temperature, osmolarity, dissolved gases
Cell line homogeneity Availability of selective media, cloning
Characterization Cytology and immuno-staining are easily performed
Preservation Can be stored in liquid nitrogen
Physiological conditions Control of hormone and nutrient concentrations

1.3 Applications of cell culture

 Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and
many biotechnology products including enzymes, hormones, immunobiologicals
(monoclonal antibodies, interleukins, lymphokines) and anticancer agents.
 Diagnosis and therapy
 Screening and studies of cell toxicity mechanisms

3
 CHAPTER 2
ANIMAL CELL CULTURE ESSENTIALS AND LAB MAINTENANCE
2.1 Cell culture lab
Any laboratory, in which tissue culture techniques are performed, regardless of the
specific purpose, must contain a number of basic facilities. These usually include the
following:
 A general washing area (Preparation area)
 Culture area

a) Washing Area
The washing area should contain large sinks, some lead-lined to resist acids and
alkalis, draining boards, and racks, and have access to demineralized water, distilled water,
and double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
baths, pipette washers and driers, and storage cabinets should also be available in the washing
area.

b) Culture Area
The most desirable arrangement is a small dust-free room equipped with an overhead
ultraviolet light and a positive-pressure ventilation unit. The ventilation should be equipped
with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of 99.97-99.99%
efficiency works well. All surfaces in the room should be designed and constructed in such a
manner that dust and microorganisms do not accumulate, and the surfaces can be thoroughly
cleaned and disinfected. A room of such design is particularly useful if large numbers of
cultures are manipulated or large pieces of equipment are utilized. Bench space for hot
plates/stirrers, pH meters, balances, water baths and media-dispensing equipment should be
available. Refrigerators and freezers for storing stock solutions and chemicals, a microwave
or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
glassware, and instruments.

2.2 Instrumentation
The specific requirements of a cell culture laboratory depend mainly on the type of
research conducted; for example, the needs of mammalian cell culture laboratory specializing

4
in cancer research is quite different from that of an insect cell culture laboratory that focuses
on protein expression. However, all cell culture laboratories have the common requirement
of being free from pathogenic microorganisms (i.e., asepsis), and share some of the same
basic equipment that is essential for culturing cells. The basic equipments are as follows:

a) CO2 incubator
Incubators are designed to promote the growth of microorganisms
or cells by maintaining a constant temperature within a narrow range.
There are many types of incubators available that offer an array of
features. Advanced features may include carbon dioxide (CO 2)
atmosphere, circulating fans, humidity controls, recording thermometers
and alarm systems. A controlled atmosphere is achieved by using a
humidifying tray and controlling the CO2 tension with a CO2-monitoring device, which draws
air from the incubator into a sample chamber, determines the concentration of CO 2, and
injects pure CO2 into the incubator to make up any deficiency. Air is circulated around the
incubator by natural convection or by using a fan to keep both the CO 2 level and the
temperature uniform. The landscape of a typical life science laboratory has changed
dramatically over the years but the CO2 incubator continues to be a staple in the research lab.
Although the ultimate goal of maintaining cell culture stocks has not changed, the functioning
and operation of CO2 incubators has become more accurate, more reliable and more
convenient.

b) Laminar flow cabinet

A laminar flow cabinet or laminar flow closet or tissue culture
hood is a carefully enclosed bench designed to prevent contamination of
semiconductor wafers, biological samples, or any particle sensitive
device. Air is drawn through a HEPA filter and blown in a very smooth,
laminar flow towards the user. The cabinet is usually made of stainless
steel with no gaps or joints where spores might collect. Laminar flow
cabinets may have a UV-C germicidal lamp to sterilize the shell and contents when not in
use. These hoods are generally available in 4-ft and 6-ft lengths, the latter being somewhat
more convenient in terms of work space. Two people can work side by side in a 6-ft hood.
This is convenient if the experimental protocol requires two people to work together. Larger

5
hoods also may be necessary if large-scale tissue culture work is to be done where many large
spinners or roller bottles are handled at the same time.
c) Inverted microscope
An inverted microscope is a microscope with its light source and condenser on the
top, above the stage pointing down, while the objectives and turret are below the stage
pointing up. Inverted microscopes are useful for observing living cells or organisms at the
bottom of a large container (e.g., a tissue culture flask) under more
natural conditions than on a glass slide which is the case with a
conventional microscope. The stage on an inverted microscope is
usually fixed, and focus is adjusted by moving the objective lens
along a vertical axis to bring it closer to or further from the specimen.
The focus mechanism typically has a dual concentric knob for coarse
and fine adjustments. Depending on the size of the microscope, four
to six objective lenses of different magnifications may be fitted to a rotating turret known as a
nosepiece. These microscopes may also be fitted with accessories for fitting still- and video
cameras, fluorescence illumination, confocal scanning and many other applications.

d) Cryocan
Cryocans are used for cryopreservation, which is a process where cells
or whole tissues are preserved by cooling to low sub-zero temperatures, such
as (typically) 77 K or −196 °C (the boiling point of liquid nitrogen). At these
low temperatures, any biological activity, including the biochemical reactions
that would lead to cell death, is effectively stopped. However, when
cryoprotectant solutions are not used, the cells being preserved are often damaged due to
freezing during the approach to low temperatures or warming to room temperature.
Phenomena which can cause damage to cells during cryopreservation mainly occur during the
freezing stage, and include solution effects, extracellular ice formation, dehydration and
intracellular ice formation. Many of these effects can be reduced by using cryoprotectants.
Most commonly used cryoprotectants are DMSO and glycerol.

6
Expanded equipment and additional supplies:

 Water bath

 Centrifuge

 Refrigerator and freezer (-20°C)

 Cell counter (e.g., Countess® Automated Cell Counter or hemocytometer)

 Sterilizer (i.e., autoclave)

 Confocal microscope

 Flow cytometer

 Water de-ionizing unit

 Fluorescent microscope

 Microplate reader

 Deep freezer (-80ºC)

 Micropipettes and/or multi-channel pipettes

 pH meter

 Microgram balance (Electronic balance)

 Lab oven

 AGE and PAGE apparatus and accessories

 Aspiration pump (peristaltic or vacuum)

7
  Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multi-well plates)

 Syringes and needles

 Waste containers

2.3 Aseptic technique and good cell culture practice

This is to ensure that all cell culture procedures are performed to a standard that will
prevent contamination from bacteria, fungi and mycoplasma, and cross contamination with
other cell lines.
 Sanitize the cabinet using 70% ethanol every time before commencing work.
 Sanitize gloves by wiping them in 70% ethanol and allow them to air dry for 30
seconds before commencing work.
 Put all materials and equipment into the cabinet prior to starting work after sanitizing
the exterior surfaces with 70% ethanol.
 While working, do not contaminate hands or gloves by touching anything outside the
cabinet (especially face and hair). If gloves become contaminated, re-sanitize with
70% ethanol as above before proceeding.
 Discard gloves after handling contaminated cultures and at the end of all cell culture
procedures each time.
 Equipment in the cabinet or that which will be taken into the cabinet during cell
culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped
with tissue soaked with 70% ethanol prior to use.
 Movement within and immediately outside the cabinet must not be rapid. Slow
movement will allow the air within the cabinet to circulate properly.
 Speech, sneezing and coughing must be directed away from the cabinet so as not to
disrupt the airflow.
 After completing work disinfect all equipment and material before removing from the
cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry
with tissue. Dispose off tissue by autoclaving.
 Sanitize the cabinet with 10 – 30 min UV light before commencing work and after
finishing work. Warning: Plastics will crack and become brittle over time with
repeated exposure to UV light. Only some cabinets have timed UV lights. Ensure they
are not left on for extended periods.
8
2.4 Fumigation of rooms using formaldehyde
Remove all unnecessary items from the room, including those that must not be exposed to the
gas, e.g., cell cultures, sensitive electrical equipment, etc.
 Clean the room with 70% ethanol to minimize the level of microbial contamination
and to allow good gas penetration
 Turn off air-handling system (roof HEPA filter system) and close the room as far as
possible and turn on the air conditioner to fan mode.
 Fill the container with formalin solution (10ml/m3 of room volume).
 Place the container in middle of the room and leave the room.
 Lock the door and display safety warning notice.
 Leave for 24-72 h.
 Afterwards, turn on cabinet and /or air-handling system.
 Leave until the level of formaldehyde reaches an acceptable level.
 Alternatively ammonia solution can be placed in a bowl and kept in the room as
ammonia absorbs the formalin vapors and neutralize them.
 The room may require cleaning to remove residues of paraformaldehyde.

2.5 Washing and sterilization of reusable lab-ware

 Soak in chromic acid solution except metals or directly in detergent.
 Wash with detergent.
 Rinse with tap-water (continuously or with sequential changes).
 Rinse with distilled water (two or more times).
 Dry in hot air oven.
 Sterilize in autoclave.
 Store for use (in dedicated cupboards or racks).

9
 CHAPTER 3
MAINTANENCE AND CULTURE OF ESTABLISHED CELL LINES
The majority of the cells are derived from vertebrates. With the exception of
hematopoietic cell lines and a few others, most are anchorage-dependent and have to be
cultured on a suitable substrate that is specifically treated to allow cell adhesion and
spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for
suspension culture. Cells that are cultured in suspension can be maintained in culture flasks
that are not tissue-culture treated, but as the culture volume to surface area is increased
beyond which adequate gas exchange is hindered (usually 0.2 – 0.5 mL/cm 2), the medium
requires agitation. This agitation is usually achieved with a magnetic stirrer or rotating
spinner flasks. The difference between adherent and suspension cell cultures and their
advantages/disadvantages are as follows:

Adherent Cell Culture Suspension Cell Culture

Appropriate for most cell types, including Appropriate for cells adapted to suspension
primary cultures culture and a few other cell lines that are non-
adhesive (e.g., hematopoietic)

10
Requires periodic passaging, but allows easy Easier to passage, but requires daily cell
visual inspection under inverted microscope counts and viability determination to follow
growth patterns; culture can be diluted to
stimulate growth

Cells are dissociated enzymatically (e.g., Does not require enzymatic or mechanical
TrypLE™ Express, trypsin) or mechanically dissociation

Growth is limited by surface area, which may Growth is limited by concentration of cells in
limit product yields the medium, which allows easy scale-up

Growth is limited by concentration of cells in Can be maintained in culture vessels that are
the medium, which allows easy scale-up not tissue-culture treated, but requires
agitation (i.e., shaking or stirring) for
adequate gas exchange

Used for cytology, harvesting products Used for bulk protein production, batch
continuously, and many research applications harvesting, and many research applications

Cell line Repositories:

The culture may be established from primary cells, or established cell cultures may be
procured from commercial or non-profit suppliers (i.e., cell banks). Reputed suppliers
provide high quality cell lines that are carefully tested for their integrity and to ensure that the
culture is free from contaminants. Regardless of their source, it is to be made sure that all
new cell lines are tested for mycoplasma contamination before one begins to use them. The
mission of cell repositories focuses on the acquisition, authentication, production,
preservation, development and distribution of standard reference microorganisms, and cell
lines for research in the life sciences. The major cell repositories are as follows:
ATCC (American type Culture Collection)
ECACC (European Collection of Animal Cell Cultures)
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen - German
Collection of Microorganisms and Cell Cultures)
JCRB (Japanese Collection of Research Bioresources)
Cell line Repository in India:

11
 The repository at National Centre for Cell Science (NCCS complex, University of
Pune Campus, Ganeshkhind, Pune 411 007, Maharashtra, India. Website:
http://www.nccs.res.in/, Email: [email protected], [email protected]) is the only
repository that houses human and animal cells in India. The NCCS repository serves to
receive, identify, maintain, store, cultivate and supply animal and human cell lines and
hybridomas. The repository has procured cultures from various sources within the country
and abroad from 35 animal species. A major bulk of the cell lines stocked in the repository
has been procured from the American Type Culture Collection (ATCC) and the European
Collection of Animal Cell Cultures (ECACC).
After receiving cells from organizations such as ATCC or NCCS, the flask in which
the cells are received should be wiped thoroughly with 70% ethanol and the morphology of
the cells should be observed. The cells should also be checked for contamination or any
change in the medium. After clear observation, the cells are kept in CO 2 incubator for one
day without any disturbance. The next day, the medium is removed and the cells are sub-
cultured for further processing.

3.1 Subculture of Adherent (monolayer) Cell Lines

Adherent cell lines will grow in vitro until they have covered the surface area
available or the medium is depleted of nutrients. Prior to this point the cell lines should be
sub-cultured in order to prevent the culture dying. To subculture the cells they need to be
brought into suspension. The degree of adhesion varies from cell line to cell line but in
majority of cases proteases, e.g. trypsin, is used to release the cells from the flask.
 Eyeball the cells - View cultures using an inverted microscope to assess the degree of
confluency and confirm the absence of bacterial and fungal contaminants.
 Check the pH of the culture medium by looking at the color of the indicator, phenol
red. As a culture becomes more acid the indicator shifts from red to yellow-red to
yellow. As the culture becomes more alkaline the color shifts from red to fuchsia (red
with a purple tinge). As a generalization, cells can tolerate slight acidity better than
they can tolerate shifts in pH above pH 7.6.

12
  Cell attachment: Are most of the cells well attached and spread out? Are the floating
cells dividing cells or dying cells which may have an irregular appearance?
 Percent confluency: The growth of a culture can be estimated by following it toward
the development of a full cell sheet (confluent culture). By comparing the amount of
space covered by cells with the unoccupied spaces one can estimate percent
confluency.
 Cell shape is an important guide: Round cells in a un-crowded culture is not a good
sign unless these happen to be dividing cells. Look for doublets or dividing cells. Get
to know the effect of crowding on cell shape.
 Look for giant cells: The number of giant cells will increase as a culture ages or
declines in "well-being." The frequency of giant cells should be relatively low and
constant under uniform culture conditions.
 One of the most valuable guides in assessing the success of a "culture split" is the rate
at which the cells in the newly established cultures attach and spread out. Attachment
within an hour or two suggests that the cells have not been traumatized and that the in
vitro environment is not grossly abnormal. Longer attachment times are suggestive of
problems. Nevertheless, good cultures may result even if attachment does not occur
for four hours.
 Keep in mind that some cells will show oriented growth patterns under some
circumstances while many transformed cells, because of a lack of contact inhibition
may "pile up" especially when the culture becomes crowded. Get to recognize the
range of cells shapes and growth patterns exhibited by each cell line.

Procedure for sub-culturing of cells:

 Remove the spent medium.
 Wash the cell monolayer with 1-2 ml of PBS without Ca2+/Mg2+ (CMF-PBS).
 Pipette trypsin onto the washed cell monolayer using 1ml per 25cm 2 of surface area.
(e.g., 1 ml – T25, 3 ml T75). Rotate flask to cover the monolayer with trypsin.
 Incubate in hood for 2-10 minutes depending on the cell line. Some cells may need to
be sitting in the incubator. Too long of a period of trypsinisation, the cells will die.
Not enough, you will not transfer enough cells.
 Once the cells start to sheet and the media becomes cloudy, move on to the next step.
You may examine the cells using an inverted microscope to ensure that many (~40%)

13
 the cells are detached and floating. It may help to “slap or tap” the flasks gently to
release any remaining attached cells.
 Re-suspend the cells in a small volume of fresh serum-containing medium to
inactivate the trypsin. Disperse the cells (this is a process to disaggregate clumps or
sheets of cells) by running the suspended cells in the medium three to four times with
the tip of the pipette on the bottom corner of the flask. Be careful not to aspirate your
media into the pipette aid. If this happens, the filter must be replaced.
 Transfer the required number of cells to a new labeled flask containing pre-warmed
medium.
Key Points
 Trypsin is inactivated in the presence of serum. Therefore, it is essential to remove all
traces of serum from the culture medium by washing the monolayer of cells with PBS
without Ca2+/Mg2+
 Cells should only be exposed to trypsin/EDTA long enough to detach cells. Prolonged
exposure could damage surface receptors.
 Trypsin should be neutralized with serum prior to seeding cells into new flasks,
otherwise cells will not attach.

Sub-culture of suspension cells:

Sub-culturing suspension cells is somewhat less complicated than passaging adherent
cells. Because the cells are already suspended in growth medium, there is no need to treat
them enzymatically to detach them from the surface of the culture vessel, and the whole
process is faster and less traumatic for the cells. Replacement of growth medium is not
carried out in suspension cultures; instead, the cells are maintained by feeding them every 2
to 3 days until they reach confluency. This can be done by directly diluting the cells in the
culture flask and continue expanding them, or by withdrawing a portion of the cells from the
culture flask and diluting the remaining cells down to a seeding density appropriate for the
cell line.

3.2 Cell counting and viability assay

Hemocytometer cell count (Trypan blue staining)

14
  Thoroughly clean the hemocytometer and its cover slip and wipe both with 70%
alcohol before use.
 Place the cover slip centrally over the counting area and across the grooves. Gently
move the cover slip back and forth over the chamber to appropriate position on the
ruler area.
 Mix the cell suspension gently and add an aliquot to the trypan blue solution (100l
cell suspension: 100l dye). The dilution will depend on the cell concentration.
 Draw a sample into a micro-pipette after mixing thoroughly and the entire tip on the
pipette to rest at the junction between the counting chamber and the cover slip.
 Using a light microscope at low power, focus on the counting chamber.
 Count the viable and non-viable cells in both halves of the chamber.

Calculations:
Total number of viable cells = A x B x C x 104
Total dead cell count = A x B x D x 104
Total cell count = viable cell count + dead cell count
% Viability = Viable cell count x 100 / Total cell count
Where A = Volume of cells
B = dilution factor in trypan blue
C= mean number of unstained cells
D= mean number of dead or stained cells
104 is the conversion factor for 0.1mm3 to 1 M

3.3 Cryopreservation

15
 If a cell line can be expanded sufficiently, preservation of cells by freezing will allow
secure stocks to be maintained without aging and protect them from problems of
contamination, incubator failure, or medium and serum crises. Ideally, 1 × 10 6 × 107 cells
should be frozen in 10 ampoules, but smaller stocks can be used if a surplus is not available.
The normal procedure is to freeze a stock of one to three ampoules as soon as surplus cells
are available, then to expand remaining cultures to confirm the identity of the cells and
absence of contamination, and freeze down a seed stock of 10–20 ampoules. One ampoule,
thawed from this stock, can then be used to generate a using stock. In many cases, there may
not be sufficient doublings available to expand the stock as much as this, but it is worth
saving some as frozen stock, no matter how little, although survival will tend to decrease
below 1 × 106 cells/ml and may not be possible below 1 × 105 cells/ml.
Factors favoring good survival after freezing and thawing are:
(i) High cell density at freezing (1 × 106 × 107 cells/ml).
(ii) Presence of a preservative, such as glycerol or dimethyl sulfoxide (DMSO) at 5–
10%.
(iii)Slow cooling, 1◦C/min, down to −70◦C and then rapid transfer to a liquid nitrogen
freezer.
(iv) Rapid thawing.
(v) Slow dilution, ∼20-fold, in medium to dilute out the preservative.
(vi) Re-seeding at 2- to 5-fold the normal seeding concentration. For example, if cells
are frozen at 5 × 106 cells in 1 ml of freezing medium with 10% DMSO and then
thawed and diluted 1:20, the cell concentration will still be 2.5 × 105 cells/ml at
seeding, higher than the normal seeding concentration for most cell lines, and the
DMSO concentration will be reduced to 0.5%, which most cells will tolerate for 24 h.
(vii) Changing medium the following day (or as soon as all the cells have attached) to
remove the cryoprotectant. Where cells are more sensitive to the cryoprotectant, they
may be centrifuged after slow dilution and re-suspended in fresh medium, but this
step should be avoided if possible as centrifugation itself may be damaging to freshly
thawed cells.

Procedure:

 Once cells get 80-90% confluent, the cells are washed with PBS and trypsinized.

16
  1x106-1x107 cells/ml is taken along with 10% serum medium and cryo-protectant such
as dimethyl sulfoxide (DMSO: 5-10%) in a cryovial.

 Then the cells are kept for slow cooling, 1°C/min, down to -70°C and then the vials
are rapidly transferred to a liquid nitrogen freezer.

3.4 Resuscitation of Frozen Cell Lines

It is vital to thaw cells correctly in order to maintain the viability of the culture and
enable the culture to recover more quickly. Some cryoprotectants, such as DMSO, are toxic
above 4ºC; therefore, it is essential that cultures are thawed quickly and diluted in culture
medium to minimize the toxic effects.
 Remove ampoule from liquid nitrogen and place in a water bath at an appropriate
temperature for your cell line e.g., 37ºC for mammalian cells. Submerge only the
lower half of the ampoule. Allow to thaw until a small amount of ice remains in the
vial - usually 1-2 minutes.
 Wipe the outside of the ampoule with a tissue moistened (not excessively) with 70%
alcohol.
 Slowly, drop wise, pipette cells into pre-warmed growth medium to dilute out the
DMSO.
 Incubate cells overnight.
 Change media the next morning. Removal of DMSO is critical.
 Examine cells microscopically (phase contrast) after 24 hours and sub-culture as
necessary.

CHAPTER 4
IN VITRO CYTOTOXICITY ASSAYS

Treating cells with a cytotoxic compound can result in a variety of cell fates. The cells
may undergo necrosis, in which they lose membrane integrity and die rapidly as a result of
cell lysis. The cells can stop actively growing and dividing (a decrease in cell viability), or
the cells can activate a genetic program of controlled cell death (apoptosis). Cytotoxicity
assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound
libraries. Researchers can either look for cytotoxic compounds, if they are interested in

17
developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can
screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before
investing in their development as a pharmaceutical. Assessing cell membrane integrity is one
of the most common ways to measure cell viability and cytotoxic effects. Compounds that
have cytotoxic effects often compromise cell membrane integrity. This chapter deals with
various cytotoxic assays.

Plating out of cells for 96-well plate cytotoxic assay

 Trypsinize a sub-confluent monolayer culture, and collect the cells in growth medium
containing serum.

 Centrifuge the suspension to pellet the cells. Re-suspend the cells in growth medium,
and count them.

 Dilute the cells depending on the growth rate of the cell line and allowing 10-20 ml of
cell suspension per 96 well plate.

 Transfer the cell suspension to a boat and, with a pipette (multichannel pipette is
preferred), add 200 l of the suspension into each well of the central 10 columns of a
96-well plate, starting with column 2 and ending with column 12 and placing 0.5 – 10
x 103 cells into each well.

 Add 200 l of growth medium to the eight wells in columns 1. Column 1 will be used
to blank the plate reader.

 Incubate the plates in a humidified atmosphere at 37 C for 24 hrs, such that the cells
are in the exponential phase of growth at the time the drug / toxicant is added.

Drug / toxins addition

 Prepare a serial five-fold dilution of the cytotoxic drug / compound in growth
medium to give ten concentrations. This set of concentrations should be chosen
such that the highest concentration kills most of the cells and the lowest kills none

18
 of the cells. Once the cytotoxicity of a drug is known, a smaller range of
concentrations can be used.

96-well plate map:

1 2 3 4 5 6 7 8 9 10 11 12

Blan Control T1 T2 T3 T4 T5 T6 T7 T8 T9 T10

k

 B = Blank (Media only)

 C = Control (Untreated cells)

 T1-T10 = Dose range of drug/toxin

Procedure:
 Remove the growth medium using pipette. Take care that the tip of the pipette does
not touch the cell sheet.
 Add required volume of growth medium to the cell sheet and add medium containing
defined concentrations of the drug.
 Follow the same for solvent control.
 Add 200 l of the medium free from the drug to the control wells.
 Incubate the plates at 37C in 5% CO2 environment for required time points.

19
4.1 MTT Assay
Principle
MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is cleaved by
mitochondrial dehydrogenase of viable cells, yielding a measurable purple formazan product.
This formazan production is proportionate to the viable cell number and inversely
proportional to the degree of cytotoxicity. Formazan can be quantified
spectrophotometrically. This assay measures the antiproliferative effects of cytotoxic drugs.

Procedure
 Add 20 l of 5mg/ml of MTT to each well.
 Wrap the plates in aluminium foil and incubate in dark for 2 to 4 h at 37C.
 Remove the medium and MTT from the wells and dissolve the remaining MTT-
formazan crystals by adding 100l of DMSO to all the wells.
 Record absorbance in a micro-plate reader at 570nm (measurement) and 630nm
(reference) immediately, since the product is unstable.

Determination of IC50
The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a
compound in inhibiting biological or biochemical function. This quantitative measure
indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a
given biological process (or component of a process, i.e. an enzyme, cell, cell receptor or
microorganism) by half. In other words, it is the half maximal (50%) inhibitory concentration
(IC) of a substance (50% IC, or IC50). It is commonly used as a measure of antagonist drug
potency in pharmacological research.

Method:
 Plot a graph of the absorbance (y-axis) against the concentration of the drug (x-axis).
 The IC50 concentration is determined as the drug concentration that is required to
reduce the absorbance to half that of the control.
 The absolute value of the absorbance should be plotted so that control values may be
compared, but the data can then be converted to a percentage-inhibition curve, to
normalize a series of curves.

20
 The percentage inhibition is calculated, from the data, using the formula:

Mean OD of untreated cells (control) - Mean OD of treated cells

= x 100
Mean OD of untreated cells (control)

 The graph can be plotted with percentage of inhibition (y-axis) against the
concentration of drug (x-axis) and IC50 concentration is determined.

21
 CHAPTER 5
MORPHOLOGICAL AND OTHER MICROSCOPIC METHODS FOR
ASSESSMENT OF CELL DEATH

There are different modes of cell death caused due to toxicity and this chapter deals
with the various morphological and other microscopic methods for assessment of cell death

Morphological assessment of the cells

5.1 Acridine Orange/Ethidium Bromide (AO/EB) staining:
Principle:
Acridine orange is a vital dye and will stain both live and dead cells. Ethidium
bromide will stain only cells that have lost membrane integrity, i.e., EB will permeate only
cells which have lost membrane integrity. Live cells will appear uniformly green. Early
apoptotic cells will stain green and contain bright green dots in the nuclei as a consequence of
chromatin condensation and nuclear fragmentation. Late apoptotic cells will also incorporate
ethidium bromide and therefore stain orange but, in contrast to necrotic cells, the late
apoptotic cells will show condensed and often fragmented nuclei. Necrotic cells stain orange,
but have a nuclear morphology resembling that of viable cells, with no condensed chromatin.

Fig: Photomicrographs of AO/EB stained HEp-2 cells

incubated for 24 hrs (A) untreated control cells. (B) toxin
treated cells; arrows showing apoptotic bodies

22
Stain Preparation
Stock:-
Acridine Orange - 10 mg/ml in PBS
Ethidium Bromide – 10 mg/ml in PBS

Working Solution:-
AO - 1 µl from stock is diluted with 100 µl of PBS
EB - 1 µl from stock is diluted with 100 µl of PBS
AO/EB stain is prepared by mixing the both stains in 1:1 ratio

Procedure
Seeding:-
 Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth
medium containing serum.
 Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count
the cells using hemocytometer.
 Add 3 ml of growth media and seed around 1,00,000 cells per well in the 6 well plate.

Drug / toxins addition:-

Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).

Staining:-
 Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for
4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend
the cells with growth medium.
 Mix the cell suspension with the AO/EB stain in 1:1 ratio on a microscopic slide and
cover with glass cover slip.

23
  Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter (450–490 nm).

5.2 Hoechst 33258 Staining:

Principle:
Hoechst 33258 stains nuclei of both live and dead cells when examined by

Fig: Hoechst 33258-stained HEp-2 cancer cells for 24h (A) Control;
(B) treated cells. Arrowheads point to the cells with abnormal
nuclei, specially indicating fragmentation of nuclei/chromatin.
fluorescence microscopy. Cells are scored apoptotic if their nuclei show chromatin
condensation and marginalization of nuclear beading or other apoptotic morphology. Often,
apoptotic nuclei fragment into smaller structures.

Procedure:-
Seeding:-
 Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth
medium containing serum.

24
  Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count
the cells using hemocytometer.
 Add 3 ml of growth media in each well and seed around 1, 00,000 cells per well in the
6 well plate.

Drug / toxins addition:-

Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).
Staining:-
 Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for
4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend
the cells with growth medium.
 Mix the cell suspension with the Hoechst 33258 stain in 1:1 ratio on a microscopic
slide and cover with glass cover slip.
 Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter (377–355 nm).

5.3 JC-1 Staining

Principle
Mitochondrial membrane potential, Δψm, is an important parameter of mitochondrial
function used as an indicator of cell health. JC-1 is a lipophilic, cationic dye that can
selectively enter the mitochondria and reversibly change color from green to red as the
membrane potential increases. In healthy cells with high mitochondrial Δψm, JC-1
spontaneously forms complexes known as J-aggregates with intense red fluorescence. On the
other hand, in apoptotic or unhealthy cells with low Δψm, JC-1 remains in the monomeric
form, which shows only green fluorescence.

25
Stain preparation:
Stock: 5mg/ml
Working solution: 20 µl of stock solution dissolved in 1 ml of DMSO

Procedure:-
Seeding:-
 Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth
medium

Fig: Photomicrographs of JC-1 stained SiHa cervical cancer cells for 6 h.

(A) control cells; (B) treated cells
containing serum.

26
  Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count
the cells using hemocytometer.
 Put a cover slip inside each well of the 6 well plate then add 3 ml of growth medium
and seed around 1,00,000 cells per well in the 6 well plate.

Drug / toxins addition:-

Treat the cells present in each well of the 6 well plate (with cover slip) with IC50
concentration of the cytotoxic drug / compound prepared in growth medium. One of the wells
should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).

Procedure

 After the completion of drug treatment period incubate the cells present in the
wells which has glass cover slips with the JC 1 working solution at 37ºC for 20
minutes.
 After 20 min, take out the cover slip with the adhered cells and keep it on the glass
slide inversely.
 The directly observe the mitochondrial depolarization patterns of treated and
control cells in a fluorescent microscope fitted with a 377-355 nm filter, at 400x
magnification and then take photographs.

5.4 Annexin V – Cy3 Assay for Apoptosis

Principle:
The annexins are a group of homologous proteins that bind phospholipids in the
presence of calcium. In living cells phosphatidylserine [PS] is transported to the inner plasma
membrane leaflet by the enzyme Mg-ATP dependent aminophospho-lipid translocase.
However, during the onset of apoptosis, PS is transported to the external leaflet of the plasma
membrane. PS is then available for binding to annexin V and any of its conjugates in the
presence of Ca2+ ions.
27
Apoptotic cells can be differentiated from necrotic cells in several ways:
 Annexin-Cy3.18 (Ann-Cy3) binds to phosphatidylserine present in the outer
leaflet of the plasma membrane of cells starting the apoptotic process. The binding
is observed as red fluorescence.
 6-Carboxyfluorescein diacetate (6-CFDA) is used to measure viability. When this
non-fluorescent compound enters living cells, esterases present hydrolyze it,
producing the fluorescent compound, 6-carboxyfluorescein (6-CF). This appears
as green fluorescence.
 Cells can be incubated either with Ann-Cy3 or 6-CFDA separately, or with the
two compounds simultaneously. After labeling at room temperature, the cells are
immediately observed by fluorescent microscopy. Live cells will be labeled only
with 6-CF (green), while necrotic cells will be labeled only with Ann-Cy3 (red).
Cells in the early stage of apoptosis, however, will be labeled with both Cy3 (red)
and 6-CF (green).

Seeding:-
 Trypsinize a sub-confluent monolayer culture, and neutralize the cells with growth
medium containing serum.
 Centrifuge and collect the pellet. Re-suspend the cells in growth medium and count
the cells using hemocytometer.
 Add 3 ml of growth media in each well and seed around 1, 00,000 cells per well in the
6 well plate.

Drug / toxins addition:-

Treat the cells with IC50 concentration of the cytotoxic drug / compound prepared in
growth medium. One of the wells should be left without drug as control.
Incubate the cells according to experimental set up (for e.g., 24 or 48 hrs).

Staining:-
 Both the live and dead cells should be collected for the morphological studies. The
supernatant containing the dead cells are first collected and centrifuged (4000 rpm for

28
 4 mins) in an eppendorf. Then the remaining live cells adhering to the well surface are
trypsinized, neutralized, centrifuged and collected in the same eppendorf. Resuspend
the cells with growth medium.
 Mix the cell suspension with the Annexin stain in 1:1 ratio on a microscopic slide and
cover with glass cover slip.
 Examine at least 300 cells in a fluorescent microscope in 40x objective using
fluorescein filter.

29
 CHAPTER 6
Assessment of genotoxicity of compounds adopting alkaline single cell gel electrophoresis
and DNA laddering assay

6.1 Comet assay for DNA damage

The comet assay or single cell gel electrophoresis (SCGE) is a rapid, sensitive and
relatively simple method for detecting DNA damage at the level of individual cells.

Fig: DNA fragmentation in toxin treated SiHa cervical cancer cells as

revealed in the comet assay. Comet images of DNA double strand breaks at
12 and 24 h treatment of toxin.

Reagents
1) 1 % Normal agarose
2) 1 % Low melting agarose
3) Electrophoresis buffer (pH 13-14)
NaOH (AR) 12 g
Na2EDTA 372 mg
Make up the volume to 1 litre with distilled water.
4) Neutralization buffer (pH 7.2)
Dissolve 12.11 g Tris-Base in 250 ml of distilled water.
5) Lysis solution (pH 10)
NaCl 146.1g
Na2 EDTA 37.2 g
Tris 1.2 g
Add the above ingredients to 700 ml distilled water and dissolve. Add about 12 g
pelletized NaOH in small quantities and stir until the salts dissolve completely into the
solution. Adjust the pH of the solution to 10.0 using 0.1N HCl or NaOH and make up to 990
30
ml with distilled water, filter-sterilize and store at room temperature. Add 10 ml of Triton X -
100 (1%) to the above solution and refrigerate for 30 - 60 min prior to use.

Dyes used
Ethidium bromide
Add 5 µl ethidium bromide stock (10 mg/ml) per 100 ml gel solution for a final
concentration of 0.5 ug/ml.

Method
Processing of cells
Trypsinize the cells in T25 flask and pellet it. Wash the pellet thrice in PBS.

Electrophoresis apparatus (set up)

The apparatus used for electrophoresis is of the conventional type. However, the gels
used for the assay are different. Since the comet assay involves the microgel electrophoresis
system, gels are loaded in 18 x 18 mm area on a fully frosted microslide. Two samples, each
containing about 1000 -2000 cells, could be conveniently cast on each slide. 8 to 12 such
slides could be placed on the platform of the electrophoresis apparatus as shown in the figure
below.

Reservoir buffer Gel containing cells

() (+)

Microscope slide

31
Preparation of slides

 Drop gently the 200µl of 1% normal agarose in PBS at 65ºC on to a fully frosted
micro-slide, cover immediately with a cover slip and place over a frozen ice pack
for about 5min.

 Remove the cover slip after the gel had set. Mix the cell suspension from one
fraction with 1% low melting agarose at 37ºC in 1:3 ratio.

 Apply 100µl of this mixture quickly on top of the gel, coated over the micro-slide
and allow to solidify as before.

 Give a third coating of 100µl of 1% low melting agarose on the gel containing the
cell suspension and allow to solidify.

 Similarly, prepare the slides for each cell fraction.

Cell lysis

 After solidification of the agarose, remove the cover slips and immerse the slides
in ice-cold lysis solution at 4ºC for 16h.

 Perform all the above operations in low lighting conditions in order to avoid any
additional DNA damage.

Electrophoresis

 Place the slides horizontally in an electrophoresis tank after being removing them
from the lysis solution.

 Fill the reservoirs with electrophoresis buffer until the slides just immerse in it.

 Allow the slides to stand in the buffer for about 20 min (to allow DNA
unwinding) after which carry out the electrophoresis at 0.8v/cm for 15 min.

32
  After electrophoresis, remove the slides, wash thrice in neutralization buffer and
gently dab to dry.

 Add a few drops of the working solution of ethidium bromide on to the gel and
cover the slide with a cover slip.

 Examine the stained DNA in the cells at 200x and 400x magnifications using a
fluorescent microscope equipped with a 365nm excitation filter and a 435nm
barrier filter.

 Measure the lengths of DNA migration (comet tail) in these cells using CASP
software. Score about 60-100 comets per point.

Application of CASP software

The comets are analysed using CASP software. The images are used to estimate the
DNA content of individual nuclei and to evaluate the degree of DNA damage representing
the fraction of total DNA in the tail. Cells are assigned to five classes: 0 (<7% of the DNA in
the tail undamaged), 1 (7–15%), 2 (15–22%), 3 (22–30%) and 4 (>30%, maximally damaged)
accordingly.

6.2 ANALYSIS OF DNA FRAGMENTATION

Programmed cell death (PCD) plays a key role in developmental biology

and in maintenance of the steady state in continuously renewing tissues. DNA
fragmentation is o n e o f the criteria for apoptotic cell death. Currently, its understanding
has been mainly from gel electrophoresis of a pooled DNA extract, as PCD (Programmed
cell death) was shown to be associated with DNA fragmentation.

During apoptosis a series of reorganization takes place in the cell:

chromatin condensations, loss of cell volume and membrane blebbing are some of the
most obvious morphological changes of apoptotic cells. Although the molecular
mechanisms leading to such changes are not completely known, many of them appear to
proceed in parallel with biochemical events. DNA cleavage is believed to be brought about
by an endogenous Ca2+ and Mg2+ dependent endonuclease capable of breaking double

33
stranded DNA at internucleosomal sites. Therefore, apoptotic DNA cleavage results in
characteristic DNA fragments of oligonucleosomal size (180-200 bp). Such phenomenon,
described for the first time by Wyllie (1980)27, can be visualized by an agarose gel
electrophoresis analysis. The present protocol provides a method for qualitative
determination of DNA fragmentation.

This protocol developed by Paola bossu (1992)28 provides a method for the
separation of fragmented DNA and their analysis by agarose gel electrophoresis. In
apoptotic cells the specific DNA cleavage becomes evident in electrophoretic analysis as
atypical ladder pattern due to multiple DNA fragments. This procedure allows the release
of fragmented chromatin from nuclei, after cell lysis (due to the presence of Triton X-100 in

the TTE solution) and disruption of the nuclear structure (following Mg2+ chelation by
EDTA in the TTE solution). The addition of the salt should be able to remove histones from
DNA.

MATERIALS

Reagents

Cell suspension at 1-5x106 cells/ml in complete DMEM medium

TTE solution: TE buffer pH 7.4 (A1) with 0.2% Triton X-100 (stored at 4°C)
5M NaCl (ice-cold)
Isopropanol (ice-cold)
70% Ethanol (ice-cold)
TE buffer (pH 7.4 )
10xLoading buffer
TBE buffer for electrophoresis
Ethidium bromide solution
Electrophoresis-grade agarose
DNA molecular weight markers

Stock Solutions:

34
 Solution Preparation Storage

TE buffer 10 mMTris.Cl pH 7.4 (prepared by diluting stock RT

solution), 1 mM EDTA.

TrisCl stock Dissolved 121 g Tris base in 800 ml water, adjusted to RT

solution (1 M) desired pH with concentrated HCl, mixed and add
water to 1 liter. CAUTION: Adjusted pH of the Tris
buffer at the same temperature at which it was to be
used, as the pH varied with temperature (about 0.028
pH units per 1°C)

Loading buffer
Prepared concentrated stock solution of loading buffer RT
10x by adding the following reagents at the indicated final
concentrations: 20% Ficoll 400, 0.1 M EDTA (pH 8.0),
1% SDS, 0.25% bromophenol blue, and 0.25% xylene
cyanol (optional)

TBE buffer stock Dissolved in 800 ml of water 108 g Tris base (89 mM), RT
solution 55 g boric acid (89 mM), 40 ml, 0.5M EDTA, pH 8.0
(2mM); brought to 1 liter with water. Used dilution
1:10

Ethidium bromide Dissolved 50 mg of ethidium bromide in 100 ml of 4°C; Protected from

stock solution H2O. Used dilution 1:1000 light

Agarose gel Dissolved 1% agarose in 1x TBE buffer (in the Prepared just before
presence of 0.5g/ml ethidium bromide) by heating until use
melted

PROCEDURE

 1×105 cells/well was seeded in 6 well microtitre plate and the plates were treated
with IC50 value of our compound and incubated for 48 hours at 37C.

 Then the cells were centrifuged at 10000 rpm for 10 minutes at 4C and collected

35
 the pellet. 0.5 ml of TTE solution was added to the pellet and vortexed vigorously.
After that the tube was centrifuged at 10000rpm for 10 minutes at 4º C. Then 0.1ml of
ice-cold 5M NaCl was added and the supernatant was transferred into a new tube and
vortexed vigorously.

 0.7 ml of ice-cold isopropanol was added to the tube and vortexed vigorously.
Precipitation was allowed to proceed overnight at -20°C.

 After overnight precipitation, the tubes were centrifuged for 15 minutes at 10,000
rpm at 4°C. The supernatant was discarded and the pellet was rinsed by adding 0.5-
0.7ml of ice cold 70% ethanol. Then it was centrifuged at 10,000rpm for 10 min at
4°C.

 The supernatant was decanted by rapidly inverting tubes and the pellet was air dried
for 30 min.

 The DNA was dissolved by adding 20-50 µl of TE solution to the tube and the
DNA sample was preserved at 4°C for 1-3 days. After that, the DNA samples were
mixed with 10x loading buffer.

 The samples were run in 1% agarose gel containing ethidium bromide 0.5
mg/ml. Appropriate DNA molecular weight markers were also added separately
for reference. The electrophoresis was stopped when the dye reached about 3 cm
from the end of the gel.

 To visualize DNA, the gel was placed on a UV transilluminator and photographs of

the gel were taken. Eye and skin protection was provided for when UV was on.

36
 CHAPTER 7
MOLECULAR TECHNIQUES FOR ASSESSING TOXICITY IN VITRO
The development of molecular techniques has yielded innovative alternative tools for
understanding and demonstrating the mechanisms underlying various biological functions. It
has also shed considerable light on a number of targeted action mechanisms of various
pharmaceutical, toxicological agents and provides a guide for researchers to study the
molecular basis of the various effects caused by these agents. This chapter deals with a few
molecular techniques for assessing toxicity in vitro.

7.1 Gene Expression Studies Adopting Polymerase Chain Reaction (PCR)

Introduction
The starting template for a PCR reaction can be DNA or RNA. DNA is usually the
appropriate template for studying the genome of the cell or tissue (as in inherited genetic
diseases, somatic mutation in a tumor, or somatic rearrangement in lymphocytes) and for the
detection of DNA viruses. For information on gene expression in a cell or tissue or the
presence of genomic RNA in a retrovirus such as HIV, RNA is the appropriate template.
RNA can be better than genomic DNA for detecting structural changes in long genes, since
amplifying the spliced RNA transcript instead of the genomic sequence greatly reduces the
length of DNA to be handled without losing any of the coding regions where clinically
significant deletions may be expected.
RT-PCR combines cDNA synthesis from RNA templates with PCR to provide a
rapid, sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify
the expression of mRNA, often from a small concentration of target RNA. The template for
RT-PCR can be total RNA or poly (A) + selected RNA. RT reactions can be primed with
random primers, oligo (dT), or a gene-specific primer (GSP) using a reverse transcriptase.
RT-PCR can be carried out either in two-step or one-step formats. In two-step RT-
PCR, each step is performed under optimal conditions. cDNA synthesis is performed first in
RT buffer and one tenth of the reaction is removed for PCR. In one-step RT-PCR, reverse
transcription and PCR take place sequentially in a single tube under conditions optimized for
both RT and PCR.

One Step RT-PCR for 25μl reaction volume

37
This protocol serves as a guideline for one-step RT-PCR with a 25μl reaction volume.
Reverse transcription and PCR are carried out sequentially in the same tube. All
components required for both reactions are added during setup, and there is no
need to add additional components once the reaction has been started. The
protocol has been optimized for 1 pg – 2 μg of total RNA. Optimal reaction
conditions, such as incubation times and temperatures during PCR amplification, will
vary and need to be determined individually

Procedure
1. Thaw template RNA, primer solutions, dNTP Mix, 5x QIAGEN OneStep RT-
PCR Buffer, and RNase-free water, and place them on ice. It is important to mix
the solutions completely before use to avoid localized differences in salt concentration.

2. Prepare a master mix according to Table 1.

The master mix typically contains all the components required for RT-PCR except
the template RNA. Prepare a volume of master mix 10% greater than that required for
the total number of reactions to be performed. A negative control (without template
RNA) should be included in every experiment

3. Mix the master mix thoroughly, and dispense appropriate volumes into PCR
tubes. Mix gently, for example, by pipetting the master mix up and down a few times.

4. Add template RNA (≤2 μg/reaction) to the individual PCR tubes.

5. Program the thermal cycler according to the program described below

6. Start the RT-PCR program while PCR tubes are still on ice. Wait until the
thermal cycler has reached 50ºC. Then place the PCR tubes in the thermal cycler.

PCR Reaction Mix

Component volume/reaction Final concentration
Master Mix
RNase-free water (provided) variable ---
5x OneStep RT-PCR Buffer 5.0µl 1x
dNTPs mix (containing 10mM of each dNTP) 1.0 µl 400 μM of each dNTP

38
Primer A variable 0.6 μM†

Primer B variable 0.6 μM†

5x OneStep RT-PCR enzyme mix 1.0 µl ---
RNase Inhibitor variable 5-10 units/reaction
Template RNA added at step 4 variable 1 pg – 2 μg/reaction

Total volume 25.0 μl ---

* Contains 12.5 mM MgCl2

† A final primer concentration of 0.6 μM is optimal for most primer–template systems.

However, in some cases using other primer concentrations (i.e., 0.5–1.0μM) may
improve amplification performance

RT PCR Reaction conditions

1. Reverse transcription: 30 min 50ºC. A reverse-transcription reaction temperature
of 50ºC is recommended. However, if satisfactory results are not obtained using 50ºC,
the reaction temperature may be increased up to 60ºC
2. Initial PCR activation: 15 min 95ºC. Hot start Taq DNA is activated in this step.
Reverse transcriptases are inactivated and the cDNA template is denatured.
3. Step Cycling
Denaturation 0.5-1min at 95ºC.
Annealing 0.5-1min at 50-58ºC (< 5ºC below Tm of primers)
Extension 1min 72ºC
Number of cycles 25-40
Final extension 10min

Analysis
Analyze the PCR reaction products by agarose gel electrophoresis of a 5 µl aliquot
from the total reaction. The products should be readily visible by UV trans-illumination of
the ethidium bromide-stained gel.

39
7.2 Protein Expression Studies Adopting Western Blot
Principle
The Western blot is an analytical technique used to detect specific proteins in a given
sample of tissue homogenate or extract. It uses SDS gel electrophoresis to separate proteins
based on their size. The proteins are then transferred to a membrane (typically nitrocellulose
or PVDF), where they are detected using antibodies specific to the target protein.
Extraction of protein
Reagents
Dignam buffer (pH: 7 to 9) (25 ml)

 Hepes 10mM (59.575 mg)

 MgCl2 1.5mM (74.25 µl from 100 mg/ml stock)

 KCl 10mM (186.4 µl from 100 mg/ml stock)

 DTT 0.5mM (38.56 µl from 50 mg/ml stock)

 PMSF 0.5mM (43.54 µl from 50 mg/ml stock)

 NP40 0.1% (25 µl)

 Store at 40 C

Note: PMSF and NP40 should be added fresh to the lysis buffer.
Procedure

 The cells are collected and centrifuged.

 The supernatant is discarded and dignam lysis buffer is added to the pellet.

 The pellet is lysed by passing through a fine syringe and incubated in deep freezer
(3 hours to overnight, freeze-thaw cycle can also be done).

 Then it is thawed and centrifuged at 12,000 to 15,000 rpm at 4o C.

40
  The supernatant is collected and stored in deep freezer (-70 o C) for later use.

Estimation of protein concentration (Bradford assay)

Principle
Bradford reagent uses Coomassie blue G-250. Without protein, the solution is red-
brown and it is an acidic solution. When protein binds the pKa of the dye shifts causing the
dye to become blue. The dye is measured at 595 nm in a spectrophotometer. Bradford dye is
easy to use, fast and sensitive.

Reagents

 Bradford reagent

Dissolve 100 mg of Coomassie blue G250 in 500 ml of ethanol, add 100 ml of

85% of phosphoric acid and make volume to 1 liter with distilled water.

 0.1M PBS

 Standard protein solution

Dissolve 5 mg of BSA in 50 ml of 0.1M PO 4. This solution contains 100

µg/pm/ml
Procedure

 Take 0.1 ml of sample solution and make the volume to 1 ml with 0.1 M PBS (pH
7.5).

 Pipette appropriate aliquots of BSA containing 0-100 µg protein. Make the

volume to 1ml with 0.1 M phosphate buffer (pH 7.5) in all tubes.

 Add 5 ml of Bradford reagent to all the tubes and mix thoroughly.

 Record the absorbance at 595 nm against the reagent blank.

 Plot a standard A595 Vs µg protein in standard.

 Determine the protein content in the sample extract from the standard curve.
41
  Calculate the amount of protein per ml sample.

Poly-Acrylamide Gel Electrophoresis (PAGE) and Western Blot

Reagents
1. Acrylamide/Bis (30% T, 2.67% C)
29.2 g acrylamide and 800 mg N’N’–bis-methylene-acrylamide are dissolved in 100
ml of de-ionized water. Filter and store at 4˚C in the dark (30 days maximum).
2. 10% (w/v) SDS
10 g SDS is dissolved in 90 ml water with gentle stirring and brought to100 ml with
de-ionized water.
3. 1.5 M Tris – HCl (pH 8.8)
18.15 g Tris base is dissolved in 80 ml de-ionized water. Adjust pH to 8.8 with 6 N
HCl and bring total volume to 100 ml with de-ionized water and store at 4˚C.
4. 0.5 M Tris – HCl (pH 6.8)
6 g Tris base is dissolved in 60 ml de-ionized water. Adjust pH to 6.8 with 6 N HCl
and bring total volume to 100 ml with de-ionized water and store at 4˚C.
5. 10% APS (Fresh daily)
100 mg Ammonium persulfate dissolved in 1 ml of de-ionized water.
6. N’ N’ - Tetramethyl ethylene diamine (TEMED)
Commercially available
7. Sample buffer (SDS reducing buffer)
1.25 ml 0.5 M Tris-HCl (pH 6.8), 2.5 ml glycerol, and 2 ml 10% (w/v) SDS are
added to 0.2 ml of 0.5% (w/v) bromophenol blue and brought to total volume of 9.5 ml
with 3.55 ml de-ionized water. The buffer is stored at room temperature. 50 μl β-
mercaptoethnaol is added to 950 µl of sample buffer prior to use.
8. 10x electrophoresis buffer (pH 8.3)
30.3 g Tris base, 144.0 g glycine and 10 g SDS are dissolved in 800 ml de-ionized
water and volume is brought to 1 L with deionized water and stored at 4◦C.
Procedure
Gel formulations (10ml)
10 % running gel is prepared by mixing the reagents as shown below.
De-ionized H2O – 4.1 ml
30% degased acrylamide/bis – 3.3 ml
1.5 M Tris – HCl (pH 8.8) – 2.5 ml
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 10 % (w/v) SDS – 0.1 ml
10% APS - 0.1 ml
TEMED - 0.004 ml
5 % stacking gel is prepared by mixing the reagents as given below.
De-ionized H2O – 5.7 ml
30% degased acrylamide/bis – 1.7 ml
0.5 M Tris – HCl (pH 6.8) – 2.5 ml
10 % (w/v) SDS – 0.1 ml
10% APS - 0.05 ml
TEMED - 0.005 ml
Separation of proteins
Proteins are separated by SDS – Polyacrylamide gel electrophoresis based on their
size as described by Laemmli (1970). By heating the sample under denaturing and reducing
conditions, proteins become unfolded and coated with SDS detergent molecule, acquiring a
high net negative charge that is proportional to the length of the polypeptide chain. When
loaded on to a gel matrix and placed in an electric field (100V), the negatively charged
protein molecules migrate towards the positively charged electrode and are separated by a
molecular sieving effect. A molecular weight protein marker (β-galactosidase, ovalbumin,
carbonic anhydrase, β-lactoglobulin or lysozyme) that produces band of known size is used to
help identify proteins of interest.
Transfer of proteins
After the separation of proteins by SDS-PAGE, the separating gel is rinsed with
distilled water and equilibrated in cold transfer buffer. Transfer sandwich is assembled in the
following order from anode (+) to cathode (-).

Reagents
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Towbin buffer (for transfer)
For 250ml
Water 93.75ml
Tris (25mM) 0.76g
Glycine 3.60g
Methanol 50ml
SDS 0.094g
pH 8.2-8.4, store at 40 c
TBST (for washing)
For 500ml preparation
Tris 0.6g
Nacl 4.383g
Tweeen 20 0.25ml
pH 7.4
PBST (for washing)
PBS 500ml
Tweeen 20 0.25ml
pH 7.4
Substrate buffer
Tris 151.42mg/25ml
TAB 10mg
H2O2

Protocol

Soak the gel membrane and pads in towbin for 20-30min

Transfer at 25V (constant voltage) for 1hour

Blocking- 2 hrs at room temperature (5%milk powder in TBS)

Add primary antibody (1:3000 dilution) and incubate for 2 hrs at RT/or overnight at 40 c

44
 Wash with PBST (6 times, 5 min each)

Incubate in 20 Ab (1:3000 dilution) in 1hour at room temperature

Wash with TBST (6 times, 5 min each)

Add substrate

Add H2O2 (80ml)

The set-up is assembled in the following way:

 Positive end

 Fiber pad

 Filter paper soaked in transfer buffer

 NC Membrane

 Gel

 Filter paper soaked in transfer buffer

 Fiber pad

 Negative end

The set up is placed in the transfer apparatus filled with cold transfer buffer and
subjected to electric field.
Blocking

45
 After the transfer, the un-reacted sites on the membrane are blocked using the
blocking solution (5% skimmed milk solution or 5% BSA) to reduce the amount of non-
specific binding.

Detection
After blocking, the protein of interest is detected using primary and enzyme linked
secondary antibodies (horseradish peroxidase-conjugated mouse/rabbit secondary antibody).

Analysis
After the unbound probes are washed away, the Western blot is ready for detection of
the probes that are labeled and bound to the protein of interest.

46