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

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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, School of 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, support and 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 School of 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 best in all my endeavors.

Thank you, God, for blessing me with all that I have.

Pallavi Raj Sharma

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Abbreviations

rpm Revolutions per minuteFBS Fetal Bovine SerumP/S Penicillin-StreptomycinPBS Phosphate Buffer Saline

DMSO Dimethyl SulphoxideddH2O Double distilled waterSDS Sodium Dodecyl Sulphate

PAGE Polyacrylamide Gel ElectrophoresisOD Optical DensityAPS Ammonium Persulfate

TEMED Tetramethylethylenediamineppm Parts per millionpsi Pounds per square inch

CONTENTS3

Page no.

1. Cell Culture 1

a. Experiment 1 : Revival 3b. Experiment 2 : Splitting 3c. Experiment 3 : Cryopreservation 5d. Experiment 4 : Treatment 5

2. Western Blotting 6a. Experiment 5 : Extraction of whole cell lysates 6b. Experiment 6 : Bradford Assay 8c. Experiment 7 : SDS-PAGE 10d. Experiment 8 : Electrotransfer 12e. Experiment 9 : Immunoblotting 13

3. Cell Culture Contamination 154. Discussion 185. References 19

1. CELL CULTURE

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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.

Basal Media contains amino acids, vitamins, inorganic salts, and a carbon source such as glucose.

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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.

Experiment 1

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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.

2. The cells were washed twice, with 2ml PBS or media and swirled then discarded.

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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.

Experiment 3

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a b

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.----------------------------------------------------------------------------------------------------

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.

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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 1.42gNaH2PO4 1.8mM 0.22gNaCl 140m

M8.19g

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.

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Tris (pH 7) 10mM 4.84gNaCl 150mM 35.04gTriton X-100 1% 2.5mlEDTA 1mM 16.66ml from

15mMEGTA 1mM 1.52gNa3VO4 0.2mM 0.147gNP-40 0.5% 0.2mlProtease 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.

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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 2mlGlycerol 2mlβ –Mercaptoethanol 1mlBromophenol 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

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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 8.03Treated 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.

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Experiment 7

Aim: SDS-PAGE for whole cell lysates

Reagents required:

1. 30% acrylamide = 29g acrylamide + 1g bisacrylamide in 100ml ddH2O2. Gel running buffer (10X)

i. Tris base =29gii. Glycine =144g

iii. SDS =10gIn a final volume of 1L, pH must be 8.3

Gel Composition:

10% Resolving Gel (10ml)

ddH2O 4ml30% acrylamide mix

3.3ml

1.5M Tris (pH8.8) 2.5ml10% SDS 0.1ml10% APS 0.1mlTEMED 0.004ml

5% Stacking Gel (3ml)

ddH2O 2.1ml30% acrylamide mix

0.5ml

1.5M Tris (pH8.8) 0.38ml10% SDS 0.03ml10% APS 0.03mlTEMED 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

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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

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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 =200mliii. 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.4. The assembly was run at 80V at 4oC for 2 hours.

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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 12.114gNaCl 1M 58.44g

Dissolve in ddH2O, adjust pH to 7.5 and make up the volume to 1L.

1X Wash buffer

10X Wash Buffer 100ml0.1% Tween 20 1ml

Blocking buffer

Non fat skimmed milk (5%) 5g1X wash buffer 100ml

Principle:

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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.

Result

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42 kDa

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.

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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.

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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.

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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.

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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, 5 th

edition, Centers for Disease Control and Prevention. Dec 2009.

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