final anti screening drug 3 document

7/31/2019 Final Anti Screening Drug 3 Document

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Screening of Anticancer Activity of the Drug

1

RAIPUR INSTITUTE OF TECHNOLOGY

Mandir Hasud, Raipur-492001

A PROJECT REPORT ON

SCREENING OF ANTICANCER ACTIVITY

OF THE DRUG

A Dissertation work Submitted in partial fulfillment of the requirement for the award of

the degree of

BACHELOR OF ENGINEERING

In

BIOTECHNOLOGY

Submitted by

Ankita Soni 3121807003

Sourabh Tiwari 3121807063

Under the guidance of

Internal Guide External Guide

Dr. Tanushree Dutta Dr. Jyothsna Rao

HOD, Dept. Of Biotechnology, Director, SRB

RITEE, Raipur (C.G.) Bangalore-64


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DECLARATION

We hereby declare that the thesis entitled SCREENING OF

ANTICANCER ACTIVITY OF DRUG which is submitted herewith

for the award of degree of Bachelor of Engineering in Biotechnology to

the Raipur Institute of Technology, Raipur, is the result of the work done

by us in SRI RAGHAVENDRA BIOTECHNOLOGIS PVT. LTD.,Bangalore under the guidance of the Dr. Jyothsna. Rao, Director, SRI

RAGHAVENDRA BIOTECHNOLOGIS PVT. Ltd.

We further declare that, the results of this work have not been previously

submitted for any degree, award of prize.

Place: Bangalore Ankita Soni

Date: Sourabh Tiwari


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ACKNOWLEDGEMENT

Any achievement, be it scholastic or otherwise does not depend solely

on the individual efforts but on the guidance, encouragement and

cooperation of intellectuals, elders and friends. A number of

personalities, in their own capacities have helped us in carrying out this

project work; we would like to take this opportunity to thanks them all.

First of all we would like to thank our HOD Tanushree Dutta, RIT,

and Raipur for her help and inspiration during the tenure of our course.

It is our pleasure to express our deepest gratitude and sincere

thanks to Dr. S.G.A Rao (Chairman, SRI RAGHAVENDRA

BIOTECHNOLOGIS PVT. Ltd Bangalore) and Dr. Jyothsna Rao

(Director, SRI RAGHAVENDRA BIOTECHNOLOGIS PVT. Ltd.Bangalore) for giving us an opportunity to work in her organization.

We express our sincere thanks to Mr. Purushothama and Mr.

Veeresh for planning the project, for balancing our project with his

schedule and for teaching us several experimental and writing skills.

This task has not been possible without his guidance, involvement and

encouragement. With his help we could accomplish our task to

perfection.

Ankita Soni

Sourabh Tiwari


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CONTENT

Chapter no Matter

I Introduction

II Materials used

III Instrument used

IV Method applied

V Protocol

VI Result

VII Reference


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CHAPTER I

INTRODUCTION


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INTRODUCTION

Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically

facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue

culture commonly refers to the culture of animal cells and tissues, while the more specific term

plant tissue culture is being named for the plants.

In 1885 Wilhelm Roux removed a portion of the medullar plate of an embryonic chicken andmaintained it in a warm saline solution for several days, establishing the basic principle of tissue

culture.

In 1907 the zoologist Ross Granville Harrison demonstrated the growth of frog nerve cell

processes in a medium of clotted lymph.

In 1913, E. Steinhardt, C. Israeli, and R. A. Lambert grew vaccine virus in fragments of guinea

pig corneal tissue

Modern usage:

In modern usage, "tissue culture" generally refers to the growth of eukaryotic cells in vitro. It is

often used interchangeably withcell culture to specifically describe the in vitro culturing ofsperm donor cells.

However, "tissue culture" can also be used to refer to the culturing of tissue pieces, i.e. explantsculture or whole organs, i.e. organ culture.

It is a tool for the study of animal cell biology in vitro model of cell growth to allow a highlyselective environment which is easily manipulated (used to optimize cell signaling pathways).

Cell culture is the complex process by which cells are grown under controlled conditions. Inpractice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell

culture are closely interrelated to those of tissue culture and organ culture.

Animal cell culture became a common laboratory technique in the mid-1900s but the concept of

maintaining live cell lines separated from their original tissue source was discovered in the 19th

century.


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2. HeLa CellHeLa cell (also Hela or HeLa cell) is a cell type in an immortal cell

line used in scientific research. It is one of the oldest and most commonly used human cell lines.The line was derived from cervical cancer cells taken from Henrietta Lacks, a patient who

eventually died of her cancer on October 4, 1951. The cell line was found to be remarkably

durable and prolific as illustrated by its contamination of many other cell lines used in research.The cells were propagated by George Otto Gey when Lacks died in 1951. This was the first

human cell line to prove successful in vitro, which was a scientific achievement with profound

future benefit to medical research. Yet Gey freely donated both the cells and the tools and

processes his lab developed to any scientists who requested them, simply for the benefit of

science. Neither Lacks nor her family gave Gey permission, and at that time, permission wasneither required nor customarily sought. The cells were later commercialized, although never

patented in their original form. Then, as now, there was no requirement to inform a patient, or

their relatives, about such matters because discarded material, or material obtained duringsurgery, diagnosis or therapy, was the property of the physician and/or medical institution. This

issue and Mrs. Lacks' situation was brought up in the Supreme Court of California case ofMoore

v. Regents of the University of California. The court ruled that a person's discarded tissue andcells are not their property and can be commercialized.

Initially, the cell line was said to be named after a "Helen Lane" or "Helen Larson", in order topreserve Lacks' anonymity. Despite this attempt, her real name was used by the press within a

few years of her death. These cells are treated as cancer cells, as they are descended from a

biopsy taken from a visible lesion on the cervix as part of Mrs. Lacks' diagnosis of cancer. Adebate still continues on the classification of the cells.

HeLa cells are termed "immortal" in that they can divide an unlimited number of times in alaboratory cell culture plate as long as fundamental cell survival conditions are met (i.e. being

maintained and sustained in a suitable environment). There are many strains of HeLa cells as

they continue to evolve by being grown in cell cultures, but all HeLa cells are descended fromthe same tumor cells removed from Mrs. Lacks. It has been estimated that the total number of

HeLa cells that have been propagated in cell culture far exceeds the total number of cells that

were in Henrietta Lacks' body.

Use in research

HeLa cells were used by slakes to test the first polio vaccine in the 1950's. Since that time HeLacells have been used for "research into cancer, AIDS, the effects of radiation and toxic

substances, gene mapping, and countless other scientific pursuits". According to author Rebecca

Skloot, by 2009, "more than 60,000 scientific articles had been published about research done

onHeLa, and that number was increasing steadily at a rate of more than 300 papers each


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CHAPTER II

MATERIALS USED


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2.1 Materials:2.1.1 Reagent used -1.Fetal Bovine Serum (FBS) : Fetal bovine serum (or fetal calf serum) is the portion ofplasma remaining after coagulation of blood, during which process the plasma protein fibrinogenis converted to fibrin and remains behind in the clot

]. Fetal bovine serum comes from the blood

drawn from a bovine fetus via a closed system venipuncture at the abattoir. Fetal bovine serum isthe most widely used serum for cell culture due to being low in antibodies and containing more

growth factors, allowing for versatility in many different applications. FBS is also used in the

culturing of eukaryotic cells.

The globular protein, bovine serum albumin (BSA), is a major component of fetal bovine serum.

The rich variety of proteins in fetal bovine serum maintains cultured cells in a medium in which

they can survive, grow, and divide.

2. Saline (0.9% NaCl) : 0.9% NaCl is normal saline. Saline is mainly used during Trypsinization of cultures.

Fig. 1: Saline


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3. Trypsin -EDTATrypsin is a porcine pancreas-derived enzyme that is commonly used for the dissociation ofcells from the culture substrate. The concentration of trypsin necessary to achieve this canvary and is dependent on a variety of factors.

EDTA is a chelating agent with gentle dissociative properties that acts to increase enzymatic

efficiency by neutralizing calcium and magnesium ions. These ions enhance cell-cell adhesion

and obscure the peptide bonds on which trypsin acts.

Component Mg/Liter Mol. Wg Mol(Mm)Inorganic Salts

EDTA 2Na.2H2O 372.2000 372.2 1.00

KCL 400.00 74.55 5.37

KH2PO4 60.00 136.09 0.44

NaHCO3 350.00 84.01 4.17

NaCl 8000.00 58.44 136.89

Trypsin 1:250 2500.00 n/a n/a

Table 1: composition of Trypsin-EDTA

Why EDTA in Trypsin?

Tissue culture media contains Calcium and Magnesium ions, fetal calf serum contains proteins

that are trypsin inhibitors. Both Mg2+/Ca2+ INHIBIT TRYPSIN. The reason why we use PBS

without Ca2+/Mg2+ to wash the cells prior to trypsinisation is to reduce the concentration of

divalent cations and proteins that inhibit trypsin action. EDTA is a Calcium chelator which will

"mop" up the remaining divalent cations. If trypsin is allowed to stay in contact with the cells for

too long a time, cell viabilty will reduce. This should be the first principle of cell culture that you

learn on day one. There are only very few cells that will detach with EDTA treatment alone.

4. Media: DMEM (Dulbeccos Modified Eagles Medium) is used for the growth of MCF-7cells. Many modifications of Eagle's Medium have been developed since the original formulation

appeared in the literature. Among the most widely used of these modifications is Dulbecco's


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Modified Eagle's medium (DMEM). DMEM is a modification of Basal Medium Eagle (BME)

that contains a four-fold higher concentration of amino acids and vitamins, as well as additional

supplementary components. The original DMEM formula contains 1000 mg/L of glucose and

was first reported for culturing embryonic mouse cells. A further alteration with 4500 mg/L

glucose has proved to be optimal for cultivation of certain cell types. DMEM (Dulbeccos

Modified Eagles Medium) is a basic cell culture medium used for a broad spectrum of

applications. Through supplementation with vitamins, amino acids, cell growth factors,

cytokines, and serum, this medium can be adapted for optimal culture of certain cell types. The

medium is produced under tightly controlled manufacturing conditions using high-quality

ingredients of animal-free origin for an optimal and consistent lot-to-lot performance. DMEM is

also available with stable glutamine

Fig. DMEM


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5. Cryopreservation:

Cryopreservation 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 pointof 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.

Cryogenic storage at very low temperatures is presumed to provide an indefinite, if not near

infinite, longevity to cells although the actual shelf life is rather difficult to prove. In

experiments with dried seeds researchers found that there was noticeable variability in

deterioration when samples were kept at different frozen temperatureseven ultra cold ones.

Temperatures below the glass transition point (TG) ofpolycots water solutions (around minus136C) appear to be accepted as the range where biological activity very substantially slows

down, and minus 196C (liquid phase of liquid nitrogen) is the preferred temperature for the

storage of important specimens. While fridges, deep freezers and extra cold deep freezers, all

similar to domestic ones, are used for many items, generally the ultra cold of liquid nitrogen at -

196C is required for successful preservation of the more complex biological structures to

virtually stop all biological activity.

One of the most important early workers on the theory of cryopreservation was James

Lovelock of Gaia theory fame. Dr. Lovelock's work suggested that damage to red blood cells

during freezing was due to osmotic stresses. Lovelock in early 1950s had also suggested thatincreasing salt concentrations in a cell as it dehydrates to lose water to the external ice might

cause damages to the cell. Cryopreservation of tissue in recent times started with the freezing of

fowl sperm, which in 1957 was cryopreserved by a team of scientists in the UK led by Dr

Christopher Polge. The process moved into the human world in the 1950s with pregnancies

obtained after insemination of frozen sperm. However, the rapid immersion of the samples in

liquid nitrogen did not, for certain of these samplessuch as types of embryos, bone marrow and

stem cellsproduce the necessary viability to make them usable on thawing. Increased

understanding of the mechanism of freezing injury to cells emphasized the importance of

controlled or slow cooling to obtain maximum survival on thawing of the living cells. A

controlled rate cooling process, allowing biological samples to equilibrate to optimal physical

parameters osmotic ally in a cryoprotectant (a form of anti-freeze) before cooling in a

predetermined, controlled way proved necessary. The ability of cryoprotectants, in the early

cases glycerol, to protect cells from freezing injury was discovered accidentally. Freezing injury

has two aspectsdirect damage from the ice crystals and secondary damage cause.


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CHAPTER III

INSTRUMENTS

USED


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3.1 Instruments3.1.1. Centrifuge: A centrifuge is a piece of equipment, generally driven by an electric motor(some older models were spun by hand), that puts an object in rotation around a fixed axis,

applying a force perpendicular to the axis. The centrifuge works using the sedimentation

principle, where the centripetal acceleration causes more dense substances to separate out alongthe radial direction (the bottom of the tube). By the same token, lighter objects will tend to move

to the top (of the tube; in the rotating picture, move to the centre).

In the picture shown, the rotating unit, called the rotor, has fixed holes drilled at an angle (to thevertical). Test tubes are placed in these slots and the rotor is spun. As the centrifugal force is in

the horizontal plane and the tubes are fixed at an angle, the particles have to travel only a little

distance before they hit the wall and drop down to the bottom. These angle rotors are very

popular in the lab for routine use.

Fig. 2: Centrifuge


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3.1.2. CO2 Incubator: In biology, an incubator is a device used to grow and maintainmicrobiological cultures or cell cultures. The incubator maintains optimal temperature, humidity

and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphereinside. Incubators are essential for a lot of experimental work in cell biology, microbiology and

molecular biology and are used to culture both bacterial as well as eukaryotic cells.

The simplest incubators are insulated boxes with an adjustable heater, typically going up to 60 to

65 C (140 to 150 F), though some can go slightly higher (generally to no more than 100 C).

The most commonly used temperature both for bacteria such as the frequently used E. coli aswell as for mammalian cells is approximately 37 C, as these organisms grow well under such

conditions. For other organisms used in biological experiments, such as the budding yeast

Saccharomyces cerevisiae, a growth temperature of 30 C is optimal. More elaborate incubatorscan also include the ability to lower the temperature (via refrigeration), or the ability to control

humidity or CO2 levels. This is important in the cultivation of mammalian cells, where the

relative humidity is typically >95% and a slightly acidic pH is achieved by maintaining a CO 2

level of 5%.Most incubators include a timer; some can also be programmed to cycle through

different temperatures, humidity levels, etc. Incubators can vary in size from tabletop to units thesize of small rooms.

Fig. 3: Incubator


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3.1.3. Heamocytometer:The hemocytometer or haemocytometer is a device

originally designed for the counting of blood cells. It is now also used to count other types of

cells as well as other microscopic particles.The hemocytometer was invented by Louis-Charles

Molasses and consists of a thick glass microscope slide with a rectangular indentation that

creates a chamber. This chamber is engraved with a laser-etched grid of perpendicular lines. The

device is carefully crafted so that the area bounded by the lines is known, and the depth of the

chamber is also known. It is therefore possible to count the number of cells or particles in a

specific volume of fluid, and thereby calculate the concentration of cells in the fluid overall.

Fig. 4: Loading of Chamber


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3.1.4. Media Filter: Its a unit used to filter the media. The filter is present b/wthe 2 containers.

Fig 5: Media Filter Unit

3.1.5. Plate reader: Micro plate Readers (also known as Plate readers) are

laboratory instruments designed to detect biological, chemical or physical events of samples in

micro titer plates. They are widely used in research, drug discovery, bioassay validation, quality

control and manufacturing processes in the pharmaceutical and biotechnological industry and

academic organizations. Sample reactions can be assayed in 6-1536 well format micro titer

plates. The most common micro plate format used in academic research laboratories or clinical

diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100

and 200 L per well. Higher density micro plates (384- or 1536-well micro plates) are typically

used for screening applications, when throughput (number of samples per day processed) and

assay cost per sample become critical parameters, with a typical assay volume between 5 and 50

L per well. Common detection modes for micro plate assays are absorbance, fluorescence

intensity, luminescence, time-resolved fluorescence, and fluorescence polarization.


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Fig. 6: Micro-Plate reader

3.2 Consumables :3.2.1. Micropipettes:Pipettes are used to accurately measure and dispense small volume of

liq. The capacity of a micropipette can range from less than 1lto 1000l (1ml), while micropipettes can

measure volumes greater than 1ml.

Glass micropipette

These are used to physically interact with microscopic samples, such as in the procedures of

microinjection and patch clamping. Most micropipettes are made of borosilicate, aluminosilicateor quartz with many types and sizes of glass tubing being available. Each of these compositions

has unique properties which will determine suitable applications.
http://en.wikipedia.org/wiki/Microlitrehttp://en.wikipedia.org/wiki/Microlitrehttp://en.wikipedia.org/wiki/Microlitre


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Fig.7:Micropipettes

3.2.2. Eppendorfs: Eppendrof tubes are polypropylene tubes. There

capacity ranges from micro centrifuge eppendrof tubes of ranges from

1.5ul to 2 ml. The racks for placing eppendrof tubes are made also of

polypropylene.

Fig.8: Eppendorf tubes


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3.2.3. Petri-dishes: A Petri dish (or Petri plate or cell culture dish) is a shallow glass or plasticcylindrical lidded dish that biologists use to culture cells. It was named after German bacteriologist Julius

Richard Petri who invented it when working as an assistant to Robert Koch. Glass Petri dishes can be

reused by sterilization (for example, in an autoclave or by dry heating in a hot air oven at 160C for one

hour). For experiments where cross-contamination from one experiment to the next can become a

problem, plastic Petri dishes may have to be disposed of after one use.

Fig.9: Petri-dishes

3.2.4. Cryo- vials: Designed for storing biological, human or animal cells, at temperatures

as low as 196C (but should be used only in the gas phase of liquid nitrogen) A silicone

washer between cap and vial ensures a positive leak proof seal.. Tubes have a white marking

area, can be color coded with a CAPINSERTand are compatible with most storage systems.

Only the round bottom vials can be centrifuged and up to 17,000g. Sterilized by gamma

radiation and packaged in unique tamperproof, safety-lock bags of 100.


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Fig.10 Cryo-vials

3.2.5. 96 well Plates: A Micro titer plate (spelled Microtitre in Europe) or micro plate

is a flat plate with multiple "wells" used as small test tubes. The micro plate has become a

standard tool in analytical research and clinical diagnostic testing laboratories. A very common

usage is in the enzyme-linked immunosorbent assay (ELISA), the basis of most modern medical

diagnostic testing in humans and animals.

Fig.11: Micro titer plate.


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CHAPTER IV

METHODS USED


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4.1 Methods

4.1.1 Preparation of media:

Media are developed by tissue culturists who want to grow different

types of cells from animal and also plants. Media is available either as

ready to use 1x media or as lyophilized powder. Ready to use media as a

shelf-life of one month at 4oC.

Medium requirements: (often empirical)

A.Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2

B. Trace elements - iron, zinc, seleniumC. sugars - glucose is the most common

D. amino acids - 13 essential

E. vitamins - B, etc.

F. choline, inositol

G. serum - contains a large number of growth promoting activities

such as buffering toxic nutrients by binding them, neutralizes

trypsin and other proteases, has undefined effects on the interaction

between cells and substrate, and contains peptide hormones or

hormone-like growth factors that promote healthy growth.

H. antibiotics - although not required for cell growth, antibiotics

are often used to control the growth of bacterial and fungal

contaminants.


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All media preparation and other cell culture work must be performed in

a laminar flow hood. Before beginning your work, turn on blower for

several minutes, wipe down all surfaces with 70% ethanol, and ethanol

wash your clean hands. Use only sterile pipettes, disposable test tubesand autoclaved pipette tips for cell culture. All culture vessels, test tubes,

pipette tip boxes, stocks of sterile eppendorfs, etc. should be opened

only in the laminar flow hood. If something is opened elsewhere in the

lab by accident, you can probably assume its contaminated. If

something does become contaminated, immediately discard the

contaminated materials into the biohazard container and notify the

instructor.

4.1.2 Subculture of Adherent Cells

Trypsin-EDTA:

a. Remove medium from culture dish and wash cells in a balanced salt solutionwithout Ca++ or Mg++. Remove the wash solution.

b. Add enough trypsin-EDTA solution to cover the bottom of the culture vesse

then pour off the excess.

c. Place culture in the laminar flow hood for 2 minutes.

d. Monitor cells under microscope. Cells are beginning to detach when they aprounded.


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4.1.3 Thawing of cells:

Materials

Chilled medium

37 degree water-bath

Procedure

Chill medium in ice-bath

Thaw cells in a 37 degree water-bath until only small pieces of ice remain.

Place tube with cells immediately on ice

Transfer cells to a centrifuge tube

Wash tube with cells a couple of times to make sure that all cells have been

transferred to the new tube.Centrifugation for a couple of minutes at 1000 rpm

Remove supernatant and resuspend cells in medium

4.1.4 Total cell count:

It determines the no. of cell in cellular suspension. This is done by heamocytom

Steps

1.Take 10ul of sample in eppendorf and add 20ul of trypan blue.2.Four cells on boundaries, only cells intersecting two of thebounda

are counted.

3.Total cell is calculated by

= Average cell count x dilution factor x104x final volume


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4.1.5 Cell viability:

To determine the % of cell living.

Procedure:

1.Take 10ul of cell suspension & mix with 20ul of trypan blue in an eppentube.

2.Clean the surface of the glass slide & the semi-silver area of heamocytomby alcohol.\

3.Mix it will & load 10ul into the meter by micropipette.4.Focus the slide under a microscope & count the living & dead cells insid

L2, L3, L4 chamber.5.Calculate the % viability of cell by

= no. living cells \ no. total cells x 100.


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CHAPTER V

PROTOCOL


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5 PROTOCOL

5.1 Isolation of MNCs from Umbilical Cord:

Protocol:

1. Take 3ml of peripheral blood and dilute with saline in 1:3.

2. Layer the sample on top of 2ml of FICOLL hypaque in 15ml

centrifuge tube.

3. Centrifuge at 1000rpm for 20min.

4. Take out MNC layer by micropipettes.

5. Transfer it to a sterile centrifuge tube.

6. Wash the cells with sterile saline by spinning at 1000rpm for 10mins.

7. Resuspend cell pellet in minimum volume of media.


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5.2 Trypsinization of HeLa cells

Protocol:

1. Discard the media from the Petri dish using sterile micropipette.

2. Wash the surface of the dish 2 times with sterile saline.3. Add 200ul of Trypsin-EDTA in to dish.

4. Rotate the dish for uniform mixing and wait for 15sec.

5. Discard Trypsin-EDTA.

6. Incubate the dish for 5min at RT.

7. Add 1ml media into the dish and mix it well.

8. Transfer 500ul of cell suspension into another sterile dish.

9. Take 1.5ml of fresh media in both the dish.10. Incubate the dishes at 37

oC in CO2 for 2448 hrs.


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5.3 Cryopreservation

Protocol:

1. Remove the culture from the dish & collect in tube.

2. Go for centrifugation at 1500 rpm for 10 min.

3. Dissolve the pellet in minimum volume of media.

4. Prepare freezing mixture (contains 70% media, 20% serum, 10%

DMSO) in ice bath.

5. Add freezing mixture to the cell suspension slowly in the ice bath.

6. Keep the cryo-vial in the slow cooling device.7. Transfer the sample to long term storage device.


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5.4 Proliferation of Lymphocytes Using a Mitogen:

Protocol:

1. Add 2ml of media into a sterile culture dish.

2. Add lymphocyte cells (1 x 106) into the dish.

3. Add PHA (10ug/ml) into the mixture.

4. Incubate for three days at 37oC in 5 % CO2 incubator.

5. Observe under an inverted micro scope for the appearance of

BLAST.

Fig. BLAST formation of Lymphocytes


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5.5 Chromosomal Preparation & Karyotyping

PROTOCOL:

1. Add 0.5ml of heparinized blood to 5ml of RPMI 1640 full

medium having 0.2ml PHA in culture tube & incubate for 68-69 hrs

at 37oC in a cell culture incubator. Culture tube should stand upright

with caps closed.

2. After 68-69 hrs culture & 10ul/ml Colcemid & mix well, incubate

for 1 hr at 37oC.

3. Centrifuge at 2000 rpm for 5 min. Discard all the supernatant

leaving 0.5ml.

4. Treat the centrifuge above pellet with 0.075 M KCl, drop-by-drop

5ml, while agitating gently.

5. Incubate at 37oC for 8-10 min.

6. Centrifuge at 2000 rpm for 5 min & discard the supernatant.

7. Add a few drops of freshly prepared fixative drop wise agitating

gently, recap the tube, & invert to mix.

8. Centrifuge the cells & remove the supernatant.

9. Resuspend the cells & fix them by adding 5ml of fixative.

10. Store the cells at 20C to 8

oC overnight.

11. Prepare the slides by dropping the above cell suspension on a

grease free slide from a distance of 2 feet.12. Air dry the slide

13. Stain with Giemsa for 30 sec.

14. Observe under the microscope.


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5.6 MTT Assay:

Protocol:

1. After 24-48 hrs of addition of drug colorimetric assay is

performed.

2. Add 20ul of MTT reagent to wells already having the media &

drug.

3. Incubate the plate for 3 hrs.

4. After 3 hrs discards the MTT reagent along with the media & the

drug, & add 100ul of DMSO (to stop the reaction of MTT).

5. Keep the plate for incubation for 1 hrs.

6. After incubation pipette out the suspension from each well into

the plate reader.

7. Read the plate on the plate reader using 550nm as test wavelength

& 630 nm as the reference wavelength.

8. Record data & tabulate column.


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CHAPTER VI

RESULT


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MTT Assay:

After 48 hrs from addition of drug MTT Assay was

carried out to determine cell viability.

Conc. Of drug

In percentage

O.D at 545 nm

Media 0.487

2.5 0.449

5.0 0.444

7.5 0.424

10.0 0.417


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