This document provides a summer training report on mammalian cell culture and western blotting techniques conducted at Jawaharlal Nehru University. It describes experiments on reviving cryopreserved cells, subculturing cells, cryopreserving cells, and treating cells with different drug concentrations. It then details the process of western blotting, including extracting whole cell lysates, estimating protein concentration using the Bradford assay, running SDS-PAGE gel electrophoresis, electrotransferring proteins to a membrane, and immunoblotting. The document provides a concise overview of key cell culture and western blotting procedures.

1. 1
Mammalian Cell Culture and Western
Blotting
Summer Training Report
Submitted to
Jawaharlal Nehru University
By
Pallavi Raj Sharma
Summer School - 2015
School of Life Sciences
Jawaharlal Nehru University
New Delhi – 110067

2. 2
Acknowledgement
I thank Prof. B. C. Tripathy, Dean, SLS, for granting me the opportunity to attend
Summer School-2015 in this esteemed institution.
I am grateful to Dr. Nirala Ramchiary, Dr. Neelima Mondal and Prof. P. C. Rath
for being easily approachable for resolving our queries and organizing this
Summer School.
I would like to thank Prof. Rana. P. Singh, Schoolof Life Sciences, Jawaharlal
Nehru University, for his guidance and encouragement. I am privileged to have
worked under his supervision.
I am highly grateful for motivation, supportand hospitality of my seniors in lab. I
sincerely thank Dr. Ajay and Ms. Reenu for their extended involvement and
encouragement which made my experience in lab worth the while. I would also
like to thank Ms. Lalita, Mr. Mathan, Mr. Mohit, Ms. Nidhi, Mr. Praveen, Ms.
Saba and Mr. Vijay for their stimulating presence, problem solving and intriguing
discussions.
I would like to express my heartfelt gratitude to the entire faculty of Schoolof Life
Sciences, Jawaharlal Nehru University for the insightful lectures, which always
sparked a curiosity to know more.
Above all, I would like to thank my parents, friends and family for their support.
Their constant encouragement and faith in me has always inspired me to keep
moving forward and give my bestin all my endeavors.
Thank you, God, for blessing me with all that I have.
Pallavi RajSharma

3. 3
Abbreviations
rpm Revolutions per minute
FBS Fetal Bovine Serum
P/S Penicillin-Streptomycin
PBS PhosphateBuffer Saline
DMSO Dimethyl Sulphoxide
ddH2O Double distilled water
SDS Sodium Dodecyl Sulphate
PAGE Polyacrylamide Gel Electrophoresis
OD Optical Density
APS Ammonium Persulfate
TEMED Tetramethylethylenediamine
ppm Parts per million
psi Pounds per square inch

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CONTENTS
Page no.
1. Cell Culture 1
a. Experiment 1 : Revival 3
b. Experiment 2 : Splitting 3
c. Experiment 3 : Cryopreservation 5
d. Experiment 4 : Treatment 5
2. Western Blotting 6
a. Experiment 5 : Extraction of whole cell lysates 6
b. Experiment 6 : Bradford Assay 8
c. Experiment 7 : SDS-PAGE 10
d. Experiment 8 : Electrotransfer 12
e. Experiment 9 : Immunoblotting 13
3. Cell Culture Contamination 15
4. Discussion 18
5. References 19

5. 5
1. CELL CULTURE
Cell culture is the method of culturing or maintaining cells procured from human
tissues in vitro, i.e. in an artificial environment providing all essential nutrients and
conditions optimally. The cells can be procured either directly from a human
biopsy sample or tissue by enzymatic or mechanical methods or can be revived
from a cryo-preserved vial containing frozen cells. Cryopreservation is the process
of storing cells at -196oC in liquid nitrogen, which freezes the cells in their present
state and inactivates all metabolic processes. This initial inoculation of cells on an
artificial growth media is referred to as primary culture.
The cells are provided with the correct temperature, gaseous tension, pH and
nutritional requirements, and thus they spread all over the plate. Once the cells
achieve 80-90% confluency, they need more space and nutrients to grow further,
thus need to be subcultured or passaged. Subculturing is the process of removing
cells from the primary culture plate and introducing them into a fresh media plate.
Many secondary plates can be prepared from a single primary plate.
While subculturing, the following numerical terms need to be considered:
1. Passage number: This refers to the number of times a cell line has been
transferred to a fresh plate. The cell line quality degrades with each passage,
sometimes show genetic drift, hence there is a limit to the number of
subcultures you can carry out. In such cases, the student must cryopreserve
the cultures at a low passage number and work with one until the maximum
passage limit is reached.
2. Split ratio: Each cell line has a specific split ratio that guides us about how
many secondary plates can be derived from one primary cell culture plate.
For instance, a split ratio of 1:4 indicates the confluent primary culture cells
can be used to initiate 4 new secondary culture plates.
Media
The growth media for optimum growth of inoculated cells consists of a
standardized mixture of essential nutrients, growth factors and hormones along
with regulating the pH and the osmotic pressure of the culture.

6. 6
Basal Media contains amino acids, vitamins, inorganic salts, and a carbon source
such as glucose.
Serum is an important component of culture media as it is a source of growth and
adhesion factors, hormones, lipids and minerals which enhances the growth
considerably. In addition, serum also regulates cell membrane permeability and
serves as a carrier for lipids, enzymes, micronutrients, and trace elements into the
cell. Although addition of serum is highly beneficial for good cell growth, there are
some problems concerned with their use including high cost, problems with
standardization, specificity, variability, and unwanted effects such as stimulation or
inhibition of growth and/or cellular function on certain cell cultures. Serums can
also be a source of contamination, therefore must be checked before adding to the
media.
The growth media is thus usually supplemented with 10% serum and 1%
antibiotics.
pH
For optimal growth of cells in artificial medium, an optimum pH of 7.4 must be
maintained. pH can be checked visually by adding few drops of media on pH test
strips. Phenol red changes color with change in pH.
CO2
Buffers provide resistance against pH changes in cell cultures and help maintaining
a constant pH. The buffering is usually achieved by including an organic (e.g.,
HEPES) or CO2-bicarbonate based buffer. The pH of the medium is prone to
change with change in external CO2 as it depends on the balance between
dissolved CO2 and bicarbonate ions in the buffering system. Hence a specific
oxygen tension needs to be maintained and 4–10% CO2 is common for most cell
culture experiments. However, each medium has a recommended CO2 tension and
bicarbonate concentration to achieve the correct pH and osmolality. This CO2
tension is maintained by a CO2 incubator where the cells are kept for incubation at
37oC.
Temperature
Most human and mammalian cells are maintained at 37°C for optimal growth.

7. 7
Experiment 1
Aim: Thawing and revival of cryopreserved cells
Materials required: Cryovial containing frozen cells, growth medium, pre-
warmed to 37°C, 15ml falcon tubes, Water bath at 37°C, 70% ethanol, tissue-
culture plates
Procedure:
1. The cryovial was taken out from liquid nitrogen storage and kept in a pre-
warmed 37°C water bath.
2. The cells were allowed to thaw completely by swirling it and the vial was taken
to the biosafety cabinet.
3. 1ml of thawed cells was added to 2ml of growth media in a falcon.
4. The cell suspension was centrifuged at 1000-1200 rpm for 5–10 minutes. A
pellet of the cells was obtained, and the supernatant was discarded without
disturbing the pellet.
5. The cells were resuspended in 3ml complete growth medium and transferred to a
culture plate and kept in incubator.
Observations: The cells were successfully revived.
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Experiment 2
Aim: Splitting revived cells – Subculturing
Materials required: Complete growth medium (RPMI 1640) (900ml), FBS
(100ml) and P/S antibiotic (10ml), sterile petriplates, Trypsin, 1X PBS, pipettes,
hemocytometer
Procedure:
1. The spent media which now appears yellow-orangish due to acidity of cell
metabolites was taken out using a pipette and discarded carefully without
disturbing the adhered cells.

8. 8
2. The cells were washed twice, with 2ml PBS or media and swirled then
discarded.
3. 1ml Trypsin was added to the petriplates and incubated in the CO2 incubator at
37oC for 5 minutes.
4. The cells were observed under the microscope for detachment.
5. 2ml complete media was added to inactivate Trypsin and the detached cells
were dispersed in the fresh media by pipetting in and out a few times.
6. The falcon tube was then centrifuged at 1000-1500 rpm for 5 minutes.
7. The supernatant was discarded and the pellet obtained of the cells was
resuspended in 5ml complete media and mixed uniformly using a pipette.
8. 10ul from the cell suspension was put in a clean hemocytometer, the cover slip
was put on and observed under microscope.
9. The number of cells was counted in each 4X4 box and noted down. The
number of cells per ml was calculated by multiplying the average cell count
with 104. Likewise, calculation was made for the volume of suspension
containing 3 lakh cells.
10.The required volume was taken and complete media was added so as to make
10ml final volume.
11.1ml each from this suspension was added to fresh plates containing 8-10ml
complete media and mixed uniformly be sliding in a zig-zag fashion.
12. The plates were checked under the microscope for uniformity and then kept in
CO 2 incubator.
Observations: After approximately 24-36 hours, the sub-cultured cells were
observed to be adhered and achieved 70-80% confluence.
Fig 1. Subcultured A549 cells observed under 40X (a) and 100X (b) magnification.
a b

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Experiment 3
Aim: Cryopreservation of cells
Materials required: Cultured cells, Typsin, DMSO, FBS, cryovial
Procedure:
1. The spent media was taken out using a pipette and discarded carefully
without disturbing the adhered cells.
2. The cells were washed twice, with 2ml PBS or media and swirled then
discarded.
3. 1ml Trypsin was added to dislodge the cells and a cell suspension was
formed.
4. The cell suspension was centrifuged and the supernatant discarded.
5. The pellet was resuspended in fresh media and 10% DMSO and 20% FBS
were added in a cryovial.
6. The cryovial was stored in -20oC storage for 2 hours then transferred to -
80oC storage overnight and finally kept at -196oC in liquid nitrogen.
Observations: Cells were cryopreserved to be used in future.
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Experiment 4
Aim: Treatment of cultured cells with different concentrations of drug
Materials required: Drug, cultured cells, pipette, growth media
Procedure:
1. The cultured cells were observed under microscope for adherence.
2. The spent growth media was replaced with fresh media without disturbing
the cells.
3. The drug was diluted to different concentrations. For instance, a 5uM and
10uM concentrated drug was prepared.
4. One control plate was left intact and 200ul of 5uM was added to one plate,
while 200ul of 10uM was added to another. This was referred to as seeding
at time zero.

10. 10
5. Seeding was performed again at time periods 6 hours and 12 hours to study
the effects of the drug on the cells overtime.
6. The cultured cells were kept in incubator.
After treating the cells with the specific doses of drug, there are many ways in
which the effect of the drug can be analyzed.
One approach is to study the changes in specific proteins in the cells, for which we
can extract whole cell lysates and proceed with western blotting.
2. WESTERN BLOTTING
Western blot is used to separate and identify proteins. The separation is based on
the molecular weight of the proteins and achieved by passing them through a gel
matrix by the process of electrophoresis. These obtained gel pattern is then
transferred to a membrane, followed by incubation with the specific primary
antibody and enzyme-conjugated secondary antibody. The detection of the single
band of the specific protein which is bound to the antibodies is carried out by
taking exposure on x-ray films. The thicker the band is on the exposed film, more
is the amount of protein present (Singh N et al., 2013).
Experiment 5
Aim: Extraction of whole cell lysates
Reagents required:
 1X PBS (For 1000ml)
Na2HPO4 10mM 1.42g
NaH2PO4 1.8mM 0.22g
NaCl 140mM 8.19g
Adjust pH to 7.4, and add double distilled water to make up the volume
to 1000ml
 Cell lysis buffer: For cell lysis and solubilizing proteins. As lysis starts,
processes like proteolysis, dephosphorylation and denaturation begin which
are slowed down using protease inhibitors.

11. 11
Tris (pH 7) 10mM 4.84g
NaCl 150mM 35.04g
Triton X-100 1% 2.5ml
EDTA 1mM 16.66ml from
15mM
EGTA 1mM 1.52g
Na3VO4 0.2mM 0.147g
NP-40 0.5% 0.2ml
Protease
Inhibitor
1X From 25X stock
Theory:
Cell lysates are the most common form of sample used for western blot. All the
cellular proteins are precipitated and collected, and the process is carried out at low
temperatures to prevent denaturation of the protein samples.
Procedure:
1. After treating the cell cultures with the specific doses for pre-decided time
period, the spent media was removed and cells were washed with 6-10ml
ice-cold 1X PBS thrice. It was ensured that PBS is completely removed.
2. 200ul of cell lysis buffer was added to each plate and incubated on ice for 20
minutes.
3. The cells were scrapped and collected in sterile microcentrifuge tubes and
these tubes were kept on ice for an additional 20 minutes followed with
storage at -20o C freezer.
4. The samples were freeze-thawed thrice.
5. The samples were centrifuged at 15000 rpm for 30 minutes at 4oC.
6. The supernatant was collected which contains whole cell lysates.
Observations: The extracted whole cell lysates were stored in -20o C freezer and
followed by protein estimation by Bradford reagent.

12. 12
Experiment 6
Aim: Protein estimation by Bradford reagent
Reagents required:
 Bradford Reagent:
(i) Solution 1 = 50mg Coomassie brilliant blue G-250 dissolved in
23.75ml 95% Ethanol and 1.25ml ddH2O
(ii) Solution 2 = Phosphoric acid (50ml) = 1.7ml ddH2O + 48.3ml
phosphoric acid
(iii) Mix solutions 1 and 2 and add ddH2O to make the volume
100ml
 2X Sample Buffer
0.5M Tris Buffer(pH
6.8)
2.5ml
20% SDS 2ml
Glycerol 2ml
β –Mercaptoethanol 1ml
Bromophenol blue 4mg
Make up the volume to 10ml with ddH2O, aliquot for long term storage at -
20o C storage.
Principle:
It is important to quantitate the extracted protein because it ensures that the
samples are being compared on an equivalent basis. The Bradford assay for protein
estimation is based on the observation that the absorbance maximum for an acidic
solution of Coomassie Brilliant Blue G-250 shifts from 465nm to 595nm when
binding to a protein occurs. Both hydrophobic and ionic interactions stabilize the
anionic form of the dye, causing a visible color change. This color change is
detected by the spectrophotometer.
After quantitation, the required amount of samples is stored in sample buffer,
which contains glycerol so that the samples sink easily into the wells of the gel. A
tracking dye (bromophenol blue) is also present to track the movement of proteins

13. 13
through the gel. The sample is heated after being diluted into a loading buffer, so
that all secondary structures are broken and the proteins are completely linear and
constitute uniform negative charge per length.
Procedure:
1. The samples were thawed for an hour and a half on ice from -20o C storage.
2. In fresh eppendorfs, 800ul of ddH2O was added along with 2ul of protein
sample.
3. The spectrophotometer was initialized and 200ul Bradford reagent was
added in each eppendorf and were mixed simultaneously.
4. Absorbance of duplicate samples was taken at 595nm and their average was
calculated.
5. The average value of absorbance was divided by 2 to obtain the value of
absorbance per ul of protein sample.
6. The volume of sample to be taken to obtain equal protein content in each
was calculated and transferred to fresh eppendorfs containing equal volume
of 2X sample buffer.
7. The samples were boiled at 95oC for 5 minutes and stored at -20o C storage.
Observations:
A1
(OD)
A2
(OD)
Avg Abs/ul Amt
(ug)
For
40ug(ul)
Vol of 2X
sample buffer(ul)
Control 0.233 0.214 0.2235 0.11175 4.98 8.03 8.03
Treated
with
5uM
0.249 0.260 0.2545 0.12725 5.75 6.95 6.95
Treated
with
10uM
0.204 0.204 0.204 0.102 4.5 8.88 8.88
All samples contain equal protein content, thus estimation is required before
running SDS-PAGE for separation of protein bands.
----------------------------------------------------------------------------------------------------

14. 14
Experiment 7
Aim: SDS-PAGE for whole cell lysates
Reagents required:
1. 30% acrylamide = 29g acrylamide + 1g bisacrylamide in 100ml ddH2O
2. Gel running buffer (10X)
i. Tris base =29g
ii. Glycine =144g
iii. SDS =10g
In a final volume of 1L, pH must be 8.3
Gel Composition:
 10% Resolving Gel (10ml)
ddH2O 4ml
30% acrylamide
mix
3.3ml
1.5M Tris (pH8.8) 2.5ml
10% SDS 0.1ml
10% APS 0.1ml
TEMED 0.004ml
 5% Stacking Gel (3ml)
ddH2O 2.1ml
30% acrylamide
mix
0.5ml
1.5M Tris (pH8.8) 0.38ml
10% SDS 0.03ml
10% APS 0.03ml
TEMED 0.003ml
Principle:
The SDS-PAGE consists of two gels: stacking and separating gel which differ in
their pHs. The stacking gel is slightly acidic which facilitates the formation of
sharp bands. The lower gel, called the separating, or resolving gel, is basic (pH

15. 15
8.8), and has a higher polyacrylamide content, which allows resolving of the
protein bands according to their molecular mass. The protein samples have
acquired negative charge due to the action of SDS, thus migrate from the negative
end to the positive end.
Procedure:
1. The resolving gel was prepared and poured into the gel casting apparatus.
2. A thin layer of methanol was added above the gel to break off contact with
air.
3. When the gel solidified, methanol was removed and stacking gel was
prepared and poured.
4. After the gel solidified, the gel was kept in the running apparatus and filled
with 1X running buffer prepared by adding 100ml of 10X running buffer in
900ml of ddH2O.
5. The comb was carefully removed ensuring the wells are intact.
6. The prepared samples were loaded carefully avoiding any debris or bubbles
to enter the well.
7. 7ul of protein ladder was also loaded for reference.
8. The gel was initially run at 60V, till the samples reach the resolving-stacking
interface, then the voltage was increased to 80V and the gel was allowed to
run for 2-3 hours.
Observations:
Fig 2. SDS-PAGE running at 80V

16. 16
Experiment 8
Aim: Electrotransfer to nitrocellulose membrane
Reagents required:
1. 10X Transfer Buffer (1L)
i. Tris Base =30g
ii. Glycine =144g
Add ddH2O to make up the volume 1L.
2. 1X Transfer Buffer (1L)
i. 10X Transfer buffer =100ml
ii. Methanol =200ml
iii. ddH2O =700ml
Principle:
After separating the protein mixture, it is transferred to a nitrocellulose membrane
using an electric field oriented perpendicular to the surface of the gel which results
of movement from proteins from the gel to the membrane. The sandwich includes
a fiber pad (sponge) at each end, and filter papers to protect the gel and blotting
membrane. It is important that the gel and membrane be in close contact and the
membrane is placed between the gel and the positive electrode. This type of
transfer is called electrophoretic transfer.
Nitrocellulose membrane: A nitrocellulose membrane has a pore size of 0.45um.
Binding of proteins to the membrane is facilitated by hydrophobic interactions and
hydrogen bonding between amino acid side chains and nitro groups of the
membrane. Partial dehydration of proteins by methanol in transfer buffer ensures
lasting bond.
Procedure:
1. The transfer tank was filled with 1X transfer buffer.
2. A stack of filter papers and transfer pads were soaked in buffer.
3. A sandwich-like arrangement was put in the apparatus with nitrocellulose
membrane facing the positive pole and gel facing the negative pole.

17. 17
4. The assembly was run at 80V at 4oC for 2 hours.
Observations:
Fig 3. Obtained nitrocellulose membrane with protein bands. Pre-stained protein
ladder on extreme left for reference.
---------------------------------------------------------------------------------------------------
Experiment 9
Aim: Immunoblotting with primary and secondary antibodies.
Reagents required:
 10X Wash buffer (1L)
Tris base 100mM 12.114g
NaCl 1M 58.44g
Dissolve in ddH2O, adjust pH to 7.5 and make up the volume to 1L.
 1X Wash buffer
10X Wash Buffer 100ml
0.1% Tween 20 1ml
 Blocking buffer
Non fat skimmed milk (5%) 5g
1X wash buffer 100ml

18. 18
Principle:
Every protein antigen has a specific unique site called epitope which allows
binding of a highly specific primary antibody. This primary antibody can also have
secondary antibodies raised against it. These secondary antibodies can be
conjugated with an enzyme which changes color or show fluorescence on addition
of a particular substrate. This fluorescence is recorded in the form of exposure on
x-ray films.
Procedure:
1. The membrane was washed once in 1X wash buffer at mild shaking.
2. The membrane was cut according to the proteins we need to detect.
3. It was kept in blocking buffer and put up for shaking for 1 hour at room
temperature.
4. The membrane was incubated with primary antibody for 1 hour at room
temperature with shaking and then incubated overnight at 4oC storage.
5. The membrane was put on shaker the next day for 1 hour and washed thrice
with 1X wash buffer.
6. The membrane was incubated with secondary antibody and kept on shaker
for 1 hour.
7. It was then washed with 1X wash buffer for 5 minutes.
8. The membrane was then kept in Luminol, until faint glowing bands start
appearing.
9. The membrane was fitted into the X-ray cassettes and was taken into the
dark room.
10.X-ray sheets were exposed for specified limited time and put in developer,
then washed in tap water and dipped in developer.
11.The bands were marked before removing the membrane.
Observations: The obtained exposed x-ray sheets are called western blots which
are used to study protein expression in cells.

19. 19
Result
Fig 4: The obtained western blot contains the protein β-actin, of size 42 kDa. It is
used as a positive control and used as a reference to confirm that equal amount of
protein was loaded initially in SDS-PAGE.
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3. CELL CULTURE CONTAMINATION
The foremost requirement while setting up and working in a cell culture lab is the
maintenance of asepsis, which refers to a completely sterile environment free of all
sorts of contaminants. Contaminants might be of different physical or chemical
nature, might vary in size – they could be invisible to the naked eye, might be
destructive or seemingly benign, but in all cases they adversely affect both the use
of cell culture and the quality of research. A contamination may results in loss of
time, resources and effort and requires a complete clean-up of the lab and
surroundings to regain its sterility. (Ryan J, 2008)
Preventing contamination is thus the prime responsibility while performing
experiments in the lab or culture hoods.
Major Cell Culture Contaminants:
Bacteria, yeast, moulds, viruses, protozoa, invertebrates (insects like ants, flies,
cockroaches) and mycoplasma are some contaminants which can be found in the
following sources:
 Media: The contaminants could come from the reagents, the water or the
sera used to prepare the media. The water must be doubly or triply distilled
to ensure complete eradication of microbial contaminants. Other origins of
contaminants could be that the protocols might be misread, the media not
properly filtered or stored.
 Storage Vessels: The reagent flasks, media vessels and culture dishes must
be properly autoclaved and wiped with 70% ethanol every time before they
are used.
42 kDa

20. 20
 Incubators: The incubators might support the growth of microbiological
contaminants and hence must be routinely cleaned to ensure its sterility.
Checking for contamination
Frequent checks for contamination must be carried out routinely to ensure that the
cell cultures are not under any threat.
 A media check must be carried out before reviving a culture.
 Observe the culture plates under microscope everyday to check for any
microbial contamination.
 Always thoroughly check the media, buffer and other reagents for unusual
viscosity or fungal contamination. Discard immediately even if a slight
contamination is visible.
 UV light must be switched on for at least 20 minutes before working in the
biosafety cabinet.
 The HEPA filters must also be cleaned routinely.
 Wipe all surfaces with 70% ethanol before and after working. Clean up
immediately in case of any spillage.
 Always use autoclaved bottles, pipettes, water and PBS.
 Filter sterilize the serum(FBS) in an autoclaved unit.
 Use antibiotics in cultures for preventing microbial growth.
 Maintain general cleanliness in the lab and always wear gloves and a lab
coat while handling reagents and cultures.
 Lab must be cleaned daily and the wastes must be properly dealt with and
discarded.
 Reagents and buffers must be accurately labeled along with the
concentration and date of preparation. Old buffers must be discarded.
If despite all precautions and awareness, a consistent contamination is
observed in the cultured cells, it is high time for a complete clean up to
prevent further loss.
First the lab must be fumigated, followed by autoclaving all possible
containments in the lab and wiping all surfaces with 70% ethanol.

21. 21
FUMIGATION
Fumigation is a method of sterilization of work spaces by filling up the room with
a fumigant, usually formaldehyde vapor which is a highly toxic compound with
maximum exposure limit of 2 ppm.
Formaldehyde vapor is an extremely effective biocidal agent. It alkylates the
carboxyl, amino, hydroxyl and sulphydral groups of proteins as well as amino
groups of nucleic acid bases, thus rendering the microorganisms inactive.
Fumigation is effective at above the temperature of 20ºC and relative humidity of
65%. (Munro K et al,1999)
Prior to fumigation, all openings and windows must be sealed. Only professionals
must perform the process and the lab must be emptied before releasing the fumes.
The room must be locked, warning signs must be put up and primary protective
equipments such as masks must be used to avoid inhalation of the fumes.
AUTOCLAVING
Autoclave is used for steam sterilization of flasks, pipettes, tips, water etc so that
they are free of contamination and ready to use. It is basically a large cylindrical
steel vessel, quarterly filled with distilled water, in which all the things to be
autoclaved are put in tightly sealed autoclave bags and the autoclave lid is closed
tightly. Then it is heated, and the initially trapped steam is removed which is called
the ‘bad steam’, thus releasing the pressure. The autoclave begins heating again,
and the pressure inside is allowed to build up to 15 psi, at 120oC for 20 minutes.

22. 22
4. DISCUSSION
It is realized that maintaining a sterile aseptic lab environment is crucial in order to
save time, effort and resources. Checking for contamination must be carried out
routinely and all precautions must be sincerely taken.
Cells are grown in vitro in an artificial complete media complemented with an
environment favorable for optimal growth of cells. It is necessary to take in
account specific pH, temperature and media requirements for specific cell lines.
The drugs used in lab are basically phytochemicals, which are plant-derived
compounds with anti-cancer properties. The drugs are introduced in in-vitro
cultures in varying concentrations or in combination with other chemopreventive
or chemotherapeutic drug and the effects of drug on cells are studied.
One approach to analyze the effect of drug is to study specific proteins and
analyzing their expression in control and treated cells. This is carried out by
performing the technique – Western Blotting. The obtained blots contain bands for
selected proteins and hence inferences can be made.

23. 23
5. REFERENCES
1. Freshney, R.I. Culture of Animal Cells: A manual of basic technique, 4th
edition, Wiley-Liss, 2000, USA.
2. Mahmood T, Yang PC. Western blot: technique, theory, and trouble
shooting. N Am J Med Sci. 2012 Sep;4(9):429-34.
3. Ryan J. Understanding and Managing Cell Culture Contamination. Corning,
Inc. Technical Bulletin. 2008, USA.
4. Cell Culture Basics Handbook, Invitrogen, Gibco.
5. Munro K, Lanser J, Flower R. A comparative study of Methods to Validate
Formaldehyde Decontamination of Biological Safety Cabinets. Appl
Environ Microbiol. 1999 Feb; 65(2): 873–876.
6. Singh N, Nambiar D, Kale RK, Singh RP. Usnic acid inhibits growth and
induces cell cycle arrest and apoptosis in human lung carcinoma A549 cells.
Nutr Cancer, 2013; 65.
7. Section IV, Biosafety in Microbiological and Biomedical Laboratories, 5th
edition, Centers for Disease Control and Prevention. Dec 2009.