Techniques in cancer cell lines experimentation
In every research aspect, techniques play the most crucial role towards achieving the desired result of various experiments. Beginning from cell revival (cancer cells), to cell culture and then subculturing for different experiments, require particularly specific protocols for each step. Cell culture involves complex processes of cell isolation from their natural environment (in vivo) and subsequent growth in a controlled environment i.e., artificial condition (in vitro). As cells reach confluency, they must be subcultured or passaged. Maintaining a constantly growing culture of cells over many months is a bad practice. There always sustain the risk of an accident that may result in the loss of cells or contamination. Therefore the cells are cryopreserved for future use. These cells can be used for various detection techniques. One of the detection method used in cancer research is Western Blotting. Western Blotting is a technique that involves the separation of proteins by gel electrophoresis, then blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibody-antigen interaction and is useful for the qualitative or semi-quantitative identification of specific proteins and their molecular weight from a complex mixture. This report contains steps including gel electrophoresis of a protein sample, transfer of protein from a gel to a membrane support, and immunodetection of a target antigen. There are many protocols for each experiment for different cell lines. The techniques used herein are particularly specific for the cell lines used and has been adopted as the precise protocol (as of now) after various trials with different possible techniques. Cancer cell lines used here were procured from ATCC (American Type Culture Collection), Manassas, Virginia and NCCS (National Centre for Cell Sciences), Pune, India, former being the pivotal host for most of the cancer cell lines used herein. Cancer cell lines used: HeLa (Cervical cancer), Skmel28 (skin cancer), A375 (skin cancer), HepG2 (liver cancer), Hep3B (liver cancer), MDAMB231 (breast cancer).
|BSA||Bovine Serum Albumin|
|DMEM||Dulbecco’s Modified Eagle’s Medium|
|FBS||Fetal Bovine Serum|
|HRP||Horse Radish Peroxidase|
|MTT||3-(4, 5-dimethylthiazol-2, 5-diphenyltetrazolium)|
|PBS||Phosphate Buffered Saline|
|PMSF||Phenyl Methyl Sulfonyl Fluoride|
|SDS||Sodium Dodecyl Sulfate|
|TBS||Tris Buffered Saline|
|TBST||Tris Buffered Saline-Tween 20|
|WCL||Whole Cell Lysate|
In a human body, under normal condition, the balance between cell birth and cell death is regulated by intricate genetic system in response to growth signals, growth inhibiting signals and death signals. Every cell in the body has specific lifespan that pave way for the new cells to gradually emerge from stem cells. Cancer is an abnormal condition of the body where these genetic control mechanisms fail to regulate the normal cell proliferation. Cancerous cells show properties of high rate cell growth and proliferation. Some of the defining properties of carcinogenesis are:
· Insensitivity to antigrowth signals
· Self sufficiency in growth signals
· Limitless replicative potential
· Evasion of apoptosis
· Tissue invasion and metastasis
· Sustained angiogenesis
Cancer cells tend to invade the adjacent normal tissues around the primary site. When the cancer cells invade other parts of the body, it is termed as metastatic cancer. Sometimes cancerous cell growth may lead to tumor formation where the dead cells accumulate and begins to form lump. Tumor can be of two types:
- Benign tumor
- Malignant tumor
Benign tumor is non-invasive and is subjected to the primary site only. But malignant tumor, on the other hand, is cancerous and easily spread to other parts of the tissues.
Many factors are responsible for cancer pathogenesis and are traceable back to DNA mutations that affects normal cell growth and cause metastasis. Mutagens are the terms for agents causing DNA mutations and these mutagens when their mutation leads to cancer are called carcinogens.
Ionizing radiations: Radon gas, prolonged exposure to UV from the sun.
Chemical carcinogens: asbestos, vinyl chloride, benzene, etc.
Infectious diseases: Viruses associated with human cancers are Hepatitis B and Hepatitis C virus, Human Papilloma virus, Epstein-Barr virus, etc.
Immune system dysfunction: HIV patients are shown to have very weak immune system
Hormonal imbalances: e.g., hyper estrogenic state causes endometrial cancer.
Heredity: Occurs when there is defective tumor suppressor allele.
Staging is the determination of how far the cancer has spread. The most commonly used method is TNM system. T in TNM denotes the tumor size, and whether or not the cancer invaded the nearby tissues and organs. N signifies spread of cancer to adjacent lymph nodes and M describes whether the cancer has metastasized to other organs of the body.
Diagnosis and treatment
The most commonly used techniques in cancer diagnosis are X-rays, blood test, CT scans and endoscopy. Malignant tumors can be confirmed by the histological examination of the cancerous cells. Treatment of cancer today is carried out by different methods such as surgery, immunotherapy, chemotherapy, radiation therapy, monoclonal antibody therapy, and many other methods depending on the location and grade of the tumor, stage of the disease and the state of the patient.
- Cryovial containing the frozen cells taken from -196o C (liquid nitrogen storage) and placed into 37oC water bath.
- Quickly thawed the cells by gently swirling the vial in the 37oC water bath until there is no ice crystals left in the vial.
- The thawed vial is transferred to a laminar flow hood and using 70% ethanol, the outside of the vial is wiped with cotton.
- Desired amount of appropriate complete growth medium, pre-warmed to 37oC, was pipetted to the vial.
- The vial with the cell suspension was centrifuged at 1500rpm for 3 minutes.
- Post centrifugation, the clarity of the supernatant and precipitation of complete cell pellets were checked.
- Supernatant aseptically decanted without the cell pellet being disturbed.
- Resuspended the cells gently in complete growth medium and transferred them into suitable culture vessel.
- The culture vessel is incubated at 37oC in 5% CO2 incubator for further use in desired experiments.
- Cells cultured in appropriate culture dish in DMEM containing 10% FBS as supplement for growth factors.
- The culture plate is then incubated at 37oC in 5% CO2 incubator for required amount of time.
- In case of contamination in the cell culture, Phenol Red changes color depending on the pH shift i.e. acidic.
Acidic medium (<6.8): Yellow
Basic medium (>8.2): Bright Pink
Neutral (6.8-8.2): Red
Subculturing of Cells
Splitting and separation
- Incubated cells are taken from incubator and transferred to laminar flow hood.
- The medium is discarded from the culture flask and administered PBS-EDTA wash twice.
- Then Trypsin-EDTA was added and incubated at 37oC incubator for 4-5 minutes.
- DMEM (double the amount of Trypsin-EDTA) was added to the trypsinised cell culture.
- The solution is transferred to 1.5ml eppendorf and centrifuged at 1300rpm for 3 minutes.
- Discarded the supernatant.
- 1ml fresh DMEM with 10% FBS was added to the eppendorf.
- 10µl aliquot of cell suspension from the eppendorf is pipetted to haemocytometer and cells counted per 0.1µl (per quadrant).
- Known numbers of cells were seeded into appropriate flask and incubated at 37oC incubator for further experiments.
- Cell culture incubated overnight at 37oC was transferred to laminar flow hood.
- Medium discarded.
- Fresh medium diluted with the drug was supplied to the cells.
- For MTT Assay, the drug treated cell culture was incubated at 37oC incubator for 72 hours.
- For Western Blotting, the cell culture was trypsinised, counted the cells in haemocytometer and seeded the required number of cells in 60mm petriplate and incubated for 24 hours.
Storage of cells
- Splitted and separated the confluent culture flask.
- Cell suspension was transferred to 2ml freezing vial and centrifuged at 1300rpm for 3 minutes.
- Supernatant discarded.
- To pellet, 1ml of freezing mixture (10% DMSO + 90% FBS) was added and thoroughly mixed.
- The vial is subsequently transferred to -4oC, -20oC, -80oC and -196oC with an interval of 30 minutes, 2 hours, and overnight incubation respectively.
- -196oC (Liquid Nitrogen) is used for long term incubation.
- 10µl aliquot of the cell culture was taken and counted in haemocytometer.
- 2000 cells/well was seeded in a micro plate and incubated at 37oC in 5% CO2 incubator for an overnight period.
- Each rows of the plate were treated with different concentrations (5µg/ml, 10µg/ml, 15µg/ml, and 50µg/ml) of the drug.
- The cells were then incubated at 37oC in CO2 incubator for 72 hours.
- The medium was removed and washed with PBS-EDTA.
- Then 100µl of fresh medium containing 25µl of MTT solution (5µg/ml in PBS) was added.
- Incubated for 2 hours at 37oC in CO2 incubator.
- The cells were then solubilised with 100µl of lysis buffer (20% SDS in 50% dimethyl formamide).
- The plate was covered with aluminum foil and incubated at 37oC in CO2 incubator for 1 hour.
- Optical densities measured at 595nm in ELISA Plate Reader.
Western blotting (or protein immunoblot) is an analytical technique to detect specific proteins in a cell extract sample. It involves denaturation of proteins with detergents and other reducing agents. The protein mixture of the cell extract is mounted onto a two layer electrophoretic gel matrix (SDS-PAGE, native PAGE, 2D gel electrophoresis, isoelectric focusing, etc.) where the proteins are sorted into distinct bands according to their molecular weight (size). Then blotting or transfer of these separated proteins to a carrier membrane (e.g.) VDF, nitrocellulose or nylon) is performed in which the exact pattern of the protein bands from the gel is pasted to the carrier membrane due to charge interaction. This immunoblot can now be used for detection of the target proteins after incubating with primary and secondary antibodies. The secondary antibody is tagged with a fluorescent or radioactive labels or enzymes which upon reaction with a specific reagent imparts color or emits light which enables the detection of the target proteins. Western blotting is widely operated in clinical diagnosis such as diagnosis for HIV infection, BSE, Hepatitis B, FIV infections, etc.
1. Cell Lysate preparation
a) Cells were seeded in 60mm petridish and incubated overnight at 37oC incubator.
b) Using a rubber scrapper, the adherent cells were scraped and then washed with 1× PBS.
c) Cell suspension was pipetted to eppendorf and centrifuged at 13000rpm at 4oC for 2 minutes.
d) Supernatant discarded and to the cell pellet, added 120µl of Whole Cell Lysate (WCL) solution.
e) Incubated in ice box for 30 minutes after mixing well.
f) Vortexed the eppendorf 6 times with 5 minutes intervals each.
g) Then centrifuged the eppendorf at 13000rpm for 10 minutes at 4oC.
h) Transferred the supernatant to fresh tube on ice.
WCL solution preparation
|VOLUME: 1019µL||VOLUME: 509.5µL|
|1M Tris (pH 7.4)||20µl||10µl|
|Ap, Lp, PMSF||5µl||2.5µl|
Bradford protein assay is a colorimetric assay used to measure the total protein concentration in a sample. It is based on the due to the binding of protein with coomassie dye under acidic condition. It actually measures the presence of basic amino acids lysine, arginine and histamine which contributes to the formation of protein-dye complex.
Polyacrylamide gels are inert, cross linked structures. The pore sizes in these gels are similar to the molecular radius of many proteins. The gels are formed by the addition of a chemical initiator and catalyst (e.g. Ammonium PerSulphate and TEMED) to a solution of acrylamide and bisacrylamide. Polyacrylamide gels are ideal for electrophoretic application for many reasons. Polyacrylamide is a thermostable medium, transparent, strong, and relatively chemically inert. A typical gel consists of two sections of different densities, cast between two glass plates. The first section to be cast, known as the resolving or separating gel, is prepared from a high concentration solution of acrylamide and bisacrylamide. Isopropanol is added to the top in order to even the layer and also to prevent bubble formation. When the resolving gel is set, a second gel known as the stacking or spacer gel, prepared from a lower concentration solution of acrylamide and bisacrylamide is cast above the resolving gel. A comb is inserted between the glass plates into the unpolymerised stacking gel to create the wells into which the samples will be loaded. The comb is then carefully removed after the gel has set and the wells are rinsed by flushing with running buffer using a pipette or syringe. Tris-glycine buffering systems or electrode buffer of pH 8.3 are the most commonly used and are comprised of a stacking gel of pH 6.8 and a resolving gel of pH 8.8.
Separating gel preparation
|Lower tris (pH 8.8)||2.5ml||2.5ml||2.5ml||2.5ml|
Stacking gel preparation
|Upper tris (pH 6.8)||315µl|
The proteins of the sample are separated using gel electrophoresis. Electrophoresis separation describes a phenomenon that charged particles move towards opposite electrode under the influence of electric field. It is used to separate proteins according to their electrophoretic mobility which depends on charge, molecule size and structure of the proteins. Polyacrylamide gel is a three-dimensional mesh networks polymer composed of acrylamide and a crosslinker (methylene bis acrylamide) under the catalyzation of ammonium per sulfate. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and can be prepared with a wide range of average pore sizes. In the presence of SDS, electrophoretic mobility is mainly based on molecular weight instead of on charge and size of the proteins. SDS is an anionic detergent which could break hydrogen bond within and between molecules to unfold proteins and break up secondary and tertiary structures as denaturing agent. Strong reducing agents such a beta-mercaptoethanol and Dithiothreitol (DTT) could disrupt disulfide linkages between cysteine residues. SDS and reducing agents are applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. The sample to be analyzed is mixed with SDS. Heating the samples to at least 60oC further promotes protein denaturation and depolymerization, helping SDS to bind and enable the rod-shape formation and negative charge adherence. A bromophenol blue dye added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run. An appropriate amount of glycerol is added to increase density and accelerate the migration of sample solution. A buffer system with different pH values is applied in gel electrophoresis process. A very widespread discontinuous buffer system is the electrode buffer system of pH 8.3 that stacks at a 6.8 and resolves at pH of 8.8. As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the positive electrode (anode) in the lower chamber, the leading ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border between the Cl- of the gel buffer and the Gly- of the cathode buffer. Because of the voltage drop between the Cl- and Glycine-buffers, proteins are compressed (stacked) into micrometer thin layer-stacking gel layer. In resolving gel layer, proteins with more negative charges per unit migrate faster than those with less negative charges per unit. That is, proteins with small molecular weight migrate faster than proteins with large molecular weight. The boundary moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in the gradient gel, for every protein at a different position.
Transfer/Electroblotting of proteins
Resolved bands in SDS-PAGE were electroboltted onto a polyvinylidene fluoride (PVDF) membrane. Cut filter papers to fit the measurement of the gel, and one PVDF membrane with the same dimension. PVDF membranes were pretreated using methanol followed by 5 minutes treatment with distilled water. Then it is treated using Towbin’s buffer to make the membrane hydrophilic. After electrophoresis, both gel and PVDF membrane of same dimension equilibrated using Towbin’s buffer. Protein bands were transferred onto PVDF membrane at 40V overnight in cold room. This method of transferring protein is called Electroblotting. It involves placing a protein containing polyacrylamide gel in direct contact with a piece of PVDF or suitable protein binding support and sandwiching this between two electrodes submerged in a conducting solution when an electric field is applied, proteins move out of gel and onto the surface of membrane were the proteins become tightly attached. After transfer, membrane stained using ponceau staining to ensure that transfer has taken place. The stain washed off completely using TBST.
The uniformity and overall effectiveness of transfer of protein from gel to the membrane can be checked by staining the membrane with ponceau dyes. Ponceau is more common due to its high sensitivity and its water solubility makes it easier to subsequently destain. It can be easily reversed with water washes, facilitating subsequent immunological detection. After transferring proteins, place membrane in an incubation tray. Enough amount of ponceau stain added to cover the membrane and incubated with gentle agitation. Then the membrane was washed with distilled water until the background is clear. Then membrane destained using running distilled water. Membrane blots dried and stored at 4oC. Membrane rewetted by placing it in methanol. Then it is placed in distilled water to remove methanol.
Since membrane has high affinity for proteins and antibodies, steps have to be taken to prevent interaction between antibodies for detection and the membrane. In order to prevent antibodies from binding to the membrane non-specifically, blocking has to be done. Blocking is often made with 5% BSA or skimmed milk diluted in TBST to reduce the background. The antibody and membrane incubated together overnight in cold room in order to protect the antibodies. When antibody is prepared in BSA, the lifetime of antibody is increased and hence primary antibody is usually prepared in BSA. Rinse the membrane in TBST after the incubation of blocking.
Treatment with primary antibody
After blocking, a dilute solution of primary antibody added and incubated with the membrane under gentle agitation. Typically the solution was comprised with 5% BSA or skimmed milk with TBST. The antibody and membrane incubated together overnight in cold room in order to protect the antibodies. When antibody is prepared in BSA, the lifetime of antibody is increased and hence primary antibody is usually prepared in BSA. Wash membrane with 1× TBST for 3 times.
Treatment with secondary antibody
After removing primary antibody, the blot was washed thrice with 1× TBST to remove unbound primary antibody and then was exposed to another antibody (anti mouse HRP, anti rabbit HRP, concentration varies from 1:5000 and 1:10000). This is known as secondary antibody. The secondary antibody is usually linked to biotin or an enzyme such as Horse Radish Peroxidase (HRP). It is usually made in 5% skimmed mild solution. The membrane was incubated with the secondary antibody for 3 hours with gentle agitation in cold room. After incubation period, the membrane was washed thrice with 1× TBST. All steps are critical for reducing non-specific binding. The membrane is detected using the labeled antibody HRP, which is detected by the signal it produces corresponding to the position of the target protein. This signal is captured on a film which is usually developed in a dark room.
Detection by enhanced chemiluminescence (ECL)
The unbound secondary antibody was washed off with TBST and detected by Enhanced Chemiluminescence (ECL) method. The chemiluminescent detection methods are based on the incubation of the western blot with a substrate that will luminesce when exposed to the secondary antibody. First the ECL solution A and B were mixed. Solution A contains chemiluminescent substrate such as luminal and p-coumaric acid. Solution B contains a strong oxidizing agent such as hydrogen peroxide. 1000µl of solution A and 2µl of solution B were mixed. After mixing the solution was poured onto the blot. Here secondary antibodies are conjugated or labeled with HRP. Luminal gets oxidized in the resence of HRP and hydrogen peroxide to form an excited state product that emits light. Light produced by this enzymatic reaction can be detected by exposure to X-ray film. Proper molecular weights can be verified by comparing band sizes to the molecular weight ladder.
Immuno Histo Chemistry (IHC)
Immunohistochemistry is a test to identify specific antigens (proteins) in a thin layer of tissue section using the principle of antibodies binding specifically to antigens in biological tissues.
- A very thin layer (approx. 5µm) of PFA-fixed tissue section is mounted on a slide.
- Dipped the slide into xylene for removal of paraffin.
- Subsequently diluted the slide into 95% ethanol 70% ethanol dH2O Citrate buffer.
- Incubated the slide in citrate buffer for 15 minutes for retrieval of the antigens.
- A boundary is mounted around the tissue section with DPX.
- After the DPX is fixed, adequate amount of endogenous IgG blocker is pipetted on the tissue section and incubated for 2 hours.
- Washed the blocker and added the second blocker i.e., protein blocker and incubated for 5 minutes.
- Washed the blocker.
- Primary antibody was incubated with the tissue section for overnight period.
- PBS washed and the tissue section is incubated with secondary antibody for 1 hour.
- PBS washed.
- DAPI is incubated with the tissue section for 10 minutes.
- Fluoro mounted the tissue section and covered with cover slip.
- Observed the slide under confocal microscope.
Clonogenic assay is a test used to determine the effectiveness of a specific agent on the growth of cells. It studies the ability of single cells in a population to from colonies after being administered and incubated with an inhibiting agent. “Clonogenic” itself infers that the cells in a colony are clones of one another.
- 500-1000 cells were seeded into a 6 well plate and incubated for 2 hours.
- It is treated with drug (inhibiting agent) and then incubated for 48 hours.
- The media is replaced with fresh media after giving PBS wash.
- Incubated for one week.
- PBS washed and new media added.
- The wells are stained with 0.5% Crystal Violet and 5% Glutaraldehyde and incubated for 10 minutes.
- Wash the plate with tap water and let it dry.
- Observed in microscope in for colony formation and plotted against graph.
RESULTS AND CONCLUSIONS
Using the methodologies in the context here, various results were obtained during trials in different experiments. One experiment would wind up in one specific technique though many other protocols could be followed with lesser fidelity. For example, the MTT Assay require different drug concentration administration, varied incubation period and concentrations of reagents used, depending upon the cancer cell line in use. Many other experiments such as Clonogenic Assay, Immunohistochemistry Assay, etc. were followed up with many differences in the techniques regarding different cell lines and each cell lines ended up with a specific technique that was different from the techniques specific for other cell lines.
It can be concluded on the understanding of the techniques used herein that different cancer cell lines are adaptive to different essence of the applied protocols in experimentation and scrutiny examination of the techniques followed and hence upgradation of each step of the protocols with new emerging techniques will greatly enhance the fidelity of the research being carried out.
|10X TBS (500ml) Tris base 12.1gm, NaCl 40gm, Make upto 50ml Adjust the pH to 7.6|
|8X ELECTRODE BUFFER Tris 12gm, Glycine 57.6gm, Make upto 500ml, Adjust pH to 8.3|
|TOWBIN’S BUFFER (500ml) Tris 1.51gm, Glycine 7.6gm, Methanol 100ml, Make up with ddH2O|
|DMEM (500ml) DMEM 13.37gm, NaHCO3 3.75gm, HEPES 5.95gm, Streptomycin & Sulphate 100mg, Penicillin 500µl, dH2O 1000ml,Adjust pH to 7.4|
|PONCEAU STAIN Ponceau 0.2% w/v, Acetic acid 10% w/v, dH2O 100ml|
|1X PBS (500ml) NaCl 8gm KCl 2gm, Na2HPO4 1.44gm, KH2PO4 0.2gm, Adjust pH to 7.4 DAD|
|1X ELECTRODE BUFFER 8X Electrode buffer 50ml, Make upto 400ml Add 4ml of SDS|
|1X TBST (500ml) 10X TBS 50ml, Make upto 500ml Tween 20 2ml, Adjust pH to 8.3|
|BRADFORD REAGENT Coomassie brilliant blue 10mg 95% ethanol 5ml 85% phosphoric acid 10ml dH2O 100ml|
|LYSIS BUFFER 20%SDS in 50% DMF|
|SDS PAGE Lower gel 14% Upper gel 5.1% H2O 4ml 30% Acrylamide 3.3ml Tris (pH 8.8) 2.5ml 10% SDS 100µl 10% APS 100µL TEMED 8µL|
|5X LOADING DYE dH2O 4ml 0.5M Tris (6.8) 1ml Glycerol 0.8ml SDS (10%) 1.6ml B-mercaptoethanol 0.4ml 0.005% bromophenol blue 0.2ml|
I, first and foremost, confer my sincere gratitude to my research guide and motivator Dr. Ruby John Anto PhD, FNASc, Scientist G, for her profound support and inspiration from initiation of the project to completion. It has been my privilege being under her guidance.
I would like to take this opportunity to express my heartfelt thanks to Professor M. Radhakrishnan Pillai, Director of Rajiv Gandhi Centre for Biotechnology for his generous support in all the accommodation facilities.
It is my utmost obligation to thank in person who has played crucial roles in the completion of my project: Jannet S (Research Assistant), Dr. Liju VB, Dr. Archana PR, Dr. Padma, Shabna A, Aishwarya US, Swetha M, Mohan Shankar, Vijai V Alex.
Your personal guidance in every step utterly enhanced my motivation during the project.
I also whole heartedly extend my personal acknowledgement to my co-trainees: Vishnu, Bincy, Adithya, Bhaghya, Farida, Bismi, Sona, Arya, Sreelaxmi, Reshma, Sudhu and Anju. Your presence made my time in the lab a joyful experience.