CHAPTER-III
3.1Chemicals
Silver nitrate (AgNO3) purchased from Molychem- Mumbai, NH3 · H2O
(25% w/w, AR), NaOH, NaCl, HNO3, Ethanol, Fetal bovine serum (FBS), PenicillinStreptomycin and 2.5 µg/ml, Amphotericin-B solution, L-Glutamine, Trypsin-EDTA
solution,
Dihydroethedium
(DHE),
Dimethly
sulfoxide
(DMSO),
V-FITC
(Fluorescein isothiocyanate) / PI (Propidium Iodide, 5µM 5-Fluoro Uracil (5-FU),
PBS, Tryphan Blue 0.4%, ethanol , RNase, Paraformaldehyde (PFA), MTT were
purchased from HiMedia Labs and SISCO research labolatories, Mumbai, India.
Milli-Q water etc.,
3.2 Glassware and Apparatus
All glassware (Conical flasks, Measuring cylinders, Beakers, Petri plates and
Test tubes etc.) were purchased from Borosil, India or from Duron Schott, Germany.
All the glassware was thoroughly washed as per standard protocols before use.
Centrifuge tubes, micropipette tips were from Helini biomolecules. T25 (Teflan flask
with 25ml capacity) flasks and 96-well microplates for tissue culture were procured
from Corning®, USA.
3.3 Media and Buffers
Nutrient broth
Nutrient agar,
LB agar,
Potato dextrose agar,
Dulbecco's Modified Eagle's Medium (DMEM)-LG
Dulbecco's Modified Eagle's Medium (DMEM)-LG
Dulbecco's Modified Eagle's Medium (DMEM)/F-12
63
(DMEM media was purchased from HiMedia Labs, Mumbai, India)
Composition of media and buffers used in the research are given in the appendix or at
appropriate places
3.4 Equipments
Autoclave – Ketan, SEMI Automatic,India
Biosafty cabinet- Labtop Instruments Private Limited, Mumbai
CO2 Incubator- Thermo Scientific
EDAX- ESEM Quanta 200, FEI.
Electronic balance – Shimadzu, AUY220
ELISA plate reader – SPECTROstar Nano, BMG LABTECH, Germany
Elix, Millipore and Milli-Q water unit
Flow cytometer- MASCQuant
FTIR- Thermo Nicolet Nexus 670 spectrophotometer; Washington, USA
Horizontal Laminar air flow- Kirloskar electrodyne Pvt.Ltd, India
Hot air oven- Technico laboratory products Pvt.Ltd, Chennai
Inverted Fluorescence microscope-EVOS FL, Life Technologies, India.
Luna automated cell counter (Logos Biosystems, India)
Micro centrifuge- eppendorf.5415R
Micro pipettes 25-200 and 100-1000 µl capacity –Helini biomolecules
Multi channel micro pipette 25-200 µl capacity- Chemtron
Normal Incubator –KEMI, India
pH meter- ELICO, India
Refrigerated incubator shaker- New Brun swick Scientific, EDISON, NJ, U.S.A
SEM- JEOL 840, Japan
TEM- 200KV HRTEM from JEOL, Japan
64
UV- Vis spectrophoto meter -Thermo scientific-EVOLUTION201
XRD- X`pert PRO PAN
3.5 Bacterial culture for silver nanoparticles production
The bacterial strain Corynebacterium glutamicum ATCC13032 was obtained
from the American Type Culture Collection (ATCC. USA). The strain was
maintained at 4°C on nutrient agar slants as well as sub cultured from time to time to
regulate its viability. Corenybacterium glutamicum is a small, non-moving Grampositive soil bacterium. It is not pathogenic, does not form spores, grows quickly, has
relatively few growth requirements, has no extracellular protease secretion and used
to produce many amino acids.
3.6 Microbial Cultures to test antimicrobial sensitivity
Bacterial strains both Gram positive (Staphylococcus aureus MTCC3160) and
gram negative strains (Ecshirishia coli MTCC40, Salmonella enteric MTCC3917,
Pseudomonas aeruginosa MTCC424, Klebsiella pneumoniae MTCC3384) Shigella
flexneri MTCC1457 which were multidrug resistant strains procured from Institute of
Microbial Technology (MTCC), Chandigarh, India and Bacillus subtilis, Bacillus
flexus were isolated from native soil of Sri Krishnadevaraya University,
Anantapuram, A.P. India and the cultures were maintained at 4˚C on nutrient agar
slants. Fungal cultures (Candida albicans, Aspergillus niger, Aspergillus flavus and
Paeciliomyces variuti) obtained from Anantapuram Medical College Anantapuram,
A.P. India, cultures were maintained at 4˚C on potato dextrose agar (PDA) slants.
3.7 Cell Lines
3.7.1 Human breast cancer cell lines (MCF-7)
65
MCF-7 is mammary epithelial cells of breast cancer. MCF-7 cells are useful
for
in vitro breast cancer studies because the cell line has retained several ideal
characteristics particular to the mammary epithelium. These include the ability for
MCF-7 cells to process estrogen, in the form of estradiol, via estrogen receptors in the
cell cytoplasm. This makes the MCF-7 cell line an estrogen receptor (ER) positive
control cell line. In addition to retaining their estrogen sensitivity, MCF-7 cells are
also sensitive to cytokeratin. They are unreceptive to desmin, endothelin, GAP, and
vimentin. When grown in vitro, the cell line is capable of forming domes and the
epithelial like cells grow in monolayers.
3.7.2 Human Liver Hepatocellular Carcinoma Cell Line (HepG2)
HepG2 is the liver tissue of hepatocellular carcinoma. These cells are
epithelial in morphology. HepG2 cells are a suitable in vitro model system for the
study of polarized human hepatocytes The cells secrete a variety of major plasma
proteins, e.g., albumin, transferrin, and the acute-phase proteins fibrinogen, alpha 2macroglobulin, alpha 1-antitrypsin, transferrin, and plasminogen. They have been
grown successfully in large-scale cultivation systems. With the proper culture
conditions, HepG2 cells display robust morphological and functional differentiation
with a controllable formation of apical and basolateral cell surface domains.
3.7.3 Human Neuroblastoma Cell Line (SK-N-SH)
The cells from which neuroblastomas are thought to arise are the
postganglionic sympathetic neuroblasts of the embryonal neural crest. The neural
crest is a transitory structure that arises during the closure of the neural tube. Neural
crest cells migrate ventrally and laterally to contribute to a variety of tissues including
the peripheral nervous system, medullary cells of the adrenal gland, calcitonin
producing cells of thyroid, pigmented cells, and mesectodermal derivatives. Growth
66
and differentiation specific peptides encountered by migratory neural crest cells are
thought to influence their development and lineage specific differentiation.
3.7.4 Rat Glioma Cell Line (C6)
C6 is a rat (Rattus norvegicus) glioma cell lines and show fibroblast
Morphology.
A glioma is a type of tumor that starts in the brain or spine. It is called
a glioma because it arises from glial cells. The most common site of gliomas is the
brain. Gliomas make up ~30% of all brain and central nervous system tumors and
80% of all malignant brain tumors.
These cells express glucocorticoid receptor.
All the cell lines were procured from National Centre for Cell Science (NCCS), Pune,
India and preserved in liquid nitrogen vapor phage.
3.8 Production of C. glutamicum biomass
C. glutamicum ATTCC13032 stock culture was maintained by sub culturing at
monthly intervals and growth conditions were optimized. A starter culture was
developed by transferring one loop full of the organism from the nutrient agar to
50mL of Luria Broth (LB) (Appendix) prepared in an Erlenmeyer flask and
autoclaved at 121±1 ºC for 15minutes then, incubated at 30˚C under shaking at 150
rpm. After 24h, 10mL of the starter culture was transferred to an Erlenmeyer flask
containing 250 mL of sterile Luria broth (LB). The culture flasks were incubated for
72 hours at 30ºC with shaking at 150 rpm. After 72 hours of growth, the biomass was
harvested by centrifugation at 5000 rpm for 10 minutes and the collected cell pellet
was washed three times with deionized water to remove the culture medium and dried
for overnight in oven at 60ºC. Weight of the dried pellet was taken and dried cells
were used for silver nanoparticles production. Dried cells also analyzed for
recognition of specific types of chemical bonds or functional groups involved in
bioreduction of silver nanoparticles by Fourier Transmission Infrared Spectroscopy
67
(FTIR) before bioreduction and the values were recorded. Milli-Q water was used
throughout the experiment.
3.9 Biosynthesis of Ag nanoparticles using biomass of C. glutamicum
ATTCC13032
Freshly prepared 15ml of diamine silver was taken into each conical flask
labeled as pH 2 to 10 and the pH values were adjusted from 2 to 10 using nitric acid
(acid-to decrease the pH) and ammonium hydroxide (base- to increase the pH). C.
glutamicum dried biomass was dissolved by adding deionized water and at the
concentration of 0.5g/L biomass was added to each conical flask containing aqueous
solutions of diamine silver complex labeled with different pH values ranging from pH
2 to pH 10 and made final volume to 25ml with deionized water, flasks containing
diamine silver and the bacterial biomass was incubated at 30°C in orbital shaker at
150rpm. Control (without the diamine silver, only biomass) was also run along with
the experimental conditions. After incubation 1mL sample was taken and centrifuged
at 5000 rpm for 10 minutes then the supernatant was assayed for the determination of
silver nanoparticles formation (Zhang et al. 2005). After bioreduction the
C.
glutamicum biomass was taken out by centrifugation at 5000rpm for 10 min and dried
the pellet at 70˚C in the oven. The dried cells were analyzed for determination of
changes in chemical bonds of bacteria involved in bioreduction by Fourier
Transmission Infrared Spectroscopy (FTIR) and the values were recorded.
Concentration of silver nanoparticles formed in the supernatant was estimated using
atomic absorption spectra.
3.10 Silver nanoparticles characterization techniques
68
Silver nanoparticle characterization is necessary to establish understanding
and control of nanoparticle synthesis and applications. Characterization is done by
using a variety of different techniques, mainly drawn from materials science.
3.10.1 UV-Vis spectrophotometer Analysis
It is generally recognized that UV-Vis
Vis spectroscopy could be used to examine
the size and shape controlled nanoparticles in aqueous suspensions (Wiley et al.
2006). In the present work supernatant containing silver nanoparticles was diluted
with deionized water (1:1) and the absorption
ion was measured using a UV
UV-Vis
spectrophotometer (Thermo
Thermo scientific-EVOLUTION201)
scientific EVOLUTION201) equipped with matching
quartz cells at a resolution of 1nm from 200-800nm. Milli-Q
Q water was
wa used as a
control. The spectroscopic studies were carried out to the reaction solutions
solutions at room
temperature.
Fig. 8 Image of UV- Vis spectrophoto meter (Thermo scientific-EVOLUTION201)
scientific EVOLUTION201)
3.10.2 FOURIER TRANSMISSION INFRARED
INFRARED SPECTOSCOPY (FTIR)
analysis
69
Recognition of specific types of chemical bonds or functional groups based on
their unique absorption signatures is possible by infrared spectroscopy Chemical bond
stretching and bending is possible by absorption of energy.
In the current study, FTIR spectra were recorded for dried biomass of C.
glutamicum before and after the bioreduction. Sample was prepared by drying the
biomass at 60°C; solid biomass of C. glutamicum was milled with 0.8% potassium
bromide (KBr) to form a very fine powder. This powder was then compressed into a
thin pellet which can be analyzed. KBr is transparent in the IR. FTIR spectra were
measured by placing the pellet in the holder of Fourier transform infrared (FTIR)
spectrophotometer (Thermo Nicolet Nexus 670 spectrophotometer; Washington,
USA); the FTIR spectrum of the dried sample was recorded in the range 493.74 to
4003.9 cm–1 at a resolution of 4 cm–1.
3.10.3 Transmission Electron Microscopy (TEM)
Transmission electron microscope (TEM) is the most widely used technique to
characterize metal nanoparticles and it was employed to visualize the size and shape
of Ag nanoparticles.
70
Fig. 9 Image of TEM (200KV
200KV HRTEM from JEOL, Japan)
Japan
AgNPs samples for TEM grids were prepared by sonicating the solution for
5minutes and placing a few drops on the 300mesh carbon-coated
carbon coated copper grid and
dried for the complete evaporation of water under a lamp and operated at an
accelerating voltage of
200 kV using EM2000Fx-II,
II, Transmission Electron
Microscope, which is a 200KV HRTEM from JEOL, Japan, to characterize the
sample after usual alignment procedures. In-situ LCD Camera is used to record the
pictures.
3.10.4 X-ray
ray diffraction (XRD) measurement
X-ray
ray diffraction crystallography (XRD) technique was performed for
determination of the dimension of biologically synthesized AgNPs with h, k, l values.
For XRD analysis, the sample containing silver nanoparticles was prepared by dropcoating onto glass substrates and dried at 45ºC in a vacuum drying oven. The vacuum
vacuumdried silver nanoparticles were used for powder X-ray
X ray diffraction (XRD) analysis.
The dried sample was placed on analytical tape and X-ray
X ray diffraction patterns were
71
recorded in the scanning mode on an X`pert PRO PAN analytical instrument operated
at 40 KV and a current of 30 mA with Cu K∝radiation.
The diffraction intensities
were recorded from 5.0170° to 81.9730, in 2θ angles, and particles size (L) of the
silver was calculated using following Debye–Scherrrer’s equation.
=
0.9λ
βcos
where, λ is the wavelength of the X-ray, β is full width and half maximum and θ is the
Bragg’s angle.
3.10.5 Scanning electron microscope (SEM)
For SEM analysis our samples were performed JEOL 840 with Resolution at
20kV: 10nm which is located in material science, IISC, Bangalore. Thin films of the
sample were prepared on a carbon coated copper grid by just dropping a very small
amount of the sample on the grid, extra solution was removed using a blotting paper
and then the film on the SEM grid were allowed to dry by putting it under a mercury
lamp for 5 min. The instrument used was a JEOL 840 with Resolution at 20kV: 10nm.
72
Fig. 10 Image of SEM JEOL 840.
3.10.6 Energy dispersive X-ray Spectroscopy (EDX) analysis
Energy Dispersive Spectrometry (EDS) technique was mainly used to
determine the elemental composition of the sample. In this study it was used to
confirm that the nanoparticle suspension contains nothing but silver. The grid for
EDX analysis was prepared by placing a drop of the silver nanoparticle suspension on
a carbon- coated copper grid and allowing the water to evaporate inside a vacuum
dryer. Energy Dispersive Spectrometry (EDS) micro-analysis of the grid containing
silver nanoparticles was performed by measuring the energy and intensity distribution
of X-ray signals generated by focusing electron beam on the specimen operating at
120kV using ESEM Quanta 200, FEI, EDX instrument. The data was used to obtain
the elemental composition of the material.
3.11 Antimicrobial assay
73
Pure cultures of testing bacteria and fungi were cultured in nutrient broth and
potato dextrose agar (PDA) respectively for 24 hours at room temperature.
Bacteria: In this method, sterile Nutrient-Agar plate was prepared. Bacterial
pathogens used in the present experiment were spread over the agar plate using sterile
cotton swab. The plates were allowed to dry and a sterile well - cutter of diameter
6.0mm was used to bore wells in the agar plates. Subsequently, a 25µl of (0.025mg)
nanoparticles suspension was introduced into wells of the inoculated Nutrient – Agar
plates and then incubated at 25°C for 24 hours and measured the diameter of
inhibitory zones in mm (Lee et al. 2010).
Fungi: Antifungal activity of obtained silver nanoparticles was evaluated by well
diffusion method against Candida albicans, Aspergillus niger, Aspergillus flavus and
Paeciliomyces variuti on Potato dextrose agar (PDA). PDA plates were prepared and
the spores of fungi were spread over the agar plate using sterile cotton swab. The
plates were allowed to dry and a sterile well - cutter of diameter 6.0mm was used to
bore wells in the plates. Subsequently, a 25µl of (0.025mg) nanoparticles suspension
was introduced into wells of the inoculated PDA plates and then incubated at 25°C for
24 hours. After incubation, the zones of inhibition were measured. The assays were
performed in triplicate and the mean values were recorded (Savithramma et al. 2011).
3.12 ANIMAL CELL AND TISSUE CULTURE
3.12.1 Recovery of Cryopreserved Cells/ Thawing
The cell lines in frozen condition are reactivated by the following procedure;
The vial from the liquid nitrogen freezer was removed and thaw by gentle
agitation in a 37°C water bath (or a bath set at the normal growth temperature for that
cell line). Thawing was done rapidly until ice crystals have melted (approximately 2
74
minutes). The vial from the water bath was removed and decontaminated it by
spraying with 70% ethanol. Strict aseptic conditions were followed in biosafty cabinet
for all further manipulations. Unscrewed the top of the vial and transferred the
contents to a sterile centrifuge tube containing 10 mL complete growth medium. The
cryoprotectant agent was removed by gentle centrifugation (10 minutes at 125 × g).
Soft cell pellet was recovered carefully by discarding the supernatant and
resuspended the cells in 1 mL or 2 mL of complete growth medium. The pellet was
loosen by gentle pipetting and broken apart clumps. (If the cells normally grow as
clusters, avoid over-pipetting during resuspension). The cell suspension was
transferred into the medium in the culture vessel (T-25 flask) and mixed thoroughly
then incubated at 37°C in a humidified atmosphere of 95% air, 5% CO2 for 24 hrs.
After 24 hours the cultures were examined and sub culture as needed.
3.12.2 Sub culturing of the cell lines
T25 flask containing a fully formed monolayer of cells was brought into
biosafty cabin. Discarded the spent medium and wash the monolayer thrice with PBS.
The adherent cells were detached by Adding 5-6 drops of Trypsin-EDTA solution
(1X/0.25 %) and allow the drops to spread over the entire monolayer. After few
minute, the bottle was vigorously shaken to facilitate the cells to come off the
substratum. Once the cells start coming off, then 5ml of medium was added. Flushed
with Pasteur pipette to dislodge the cells adhering to the glass surface and incubated at
37ºC. The fresh medium was replaced for every three days to remove the dead cells
along with the spent medium.
3.12.3 Cell counting and viability
Hemocytometer was taken and placed it on the flat surface of the work bench.
Cover slip was placed on the counting chamber. 0.1 ml of 0.4% Trypan Blue dye was
75
added to
0.5ml of cell suspension in a tube. Dead cells were stained red with
Trypan Blue. Pipetting from the edge of the cover slip cell suspension was applied to
the cleaned hemocytometer and permitting diffusion by capillary action. Care was
taken that there was no air bubbles and there was no overfilling beyond the ruled area.
The counting chamber was placed on the bench for 2-3 minutes to allow the cells to
settle. Counting chamber of the hemocytometer was placed on the stage of the
microscope between the clips to hold the slide so that the counting chamber can be
moved (if the microscope is provided with a moving stage). Switched to low power
(10x) objective, adjusted the light (less light needed, hence close the aperture or lower
the condenser) and focussed on the wall of the counting chamber. Then slowly
moving the stage towards the middle of the slide until the ruling area visible, sharpen
the focus and located the large square in the centre.
The number of unstained cells were counted seen on the small square
(0.2x0.2=0.04sq mm) of the upper left corner which was divided into 16 smaller
squares to facilitate counting. Counting was repeated with three other corner squares.
Total of all the cells counted in
4 squares was made. Repeated the same on the
other side of the chamber and an average of the two chambers was calculated.
The concentration of cells in the original suspension in cells/ml =
No.ofcellscounted
No.ofGridscounted
76
× 104 × 2
Fig. 11 Image of Inverted microscope (EVOS FL, Life Technologies, India)
3.12.4 Preservation of cells
During Preservation metabolic activity of the cells get inactivated or rate of cell
division get slow by the addition of cryoprotectant i.e. Dimethyl sulfoxide (DMSO)
and Glycerol in the appropriate concentration with liquid nitrogen this type of
preservation is called cryopreservation. With which cells will be alive for many
number of years in viable condition for future works.
Freeze medium was prepared by adding 5% DMSO to complete growth
medium.
(Do not add undiluted DMSO to a cell suspension as dissolution of
DMSO in aqueous solutions gives off heat). T25 flask containing a fully formed
monolayer of cells was brought into biosafty cabin. Discarded the spent medium and
washed the monolayer thrice with PBS. The adherent cells were detached by adding
5-6 drops of Trypsin-EDTA solution
(1X/0.25 %) and allowed the drops to spread
over the entire monolayer. After few minute, the bottle was vigorously shaken to
77
facilitate the cells to come off the substratum. Once the cells started to coming off,
and then added 5ml of medium. Cells adhering to the glass surface were dislodged by
flushing the cells with Pasteur pipette. Cells were collected by gentle centrifugation (5
minutes at 2000rpm) and the cell pellet was resuspended with 5ml DMEM media.
The freeze medium at a concentration of 1:1 was added to the cells. The vials were
labeled with appropriate number, the name of the cell line and the date. The vials
were placed into a pre-cooled (4°C), controlled-rate freeze chamber and placed the
chamber in a mechanical freezer at –80°C (or colder) for at least 24 hours. The vials
were transferred quickly to liquid nitrogen or –130°C freezers. The location and
details of the freeze was recorded and the cells were kept at –130°C till we use. To
recover the cells from the freezer, thaw the cells rapidly at 37ºC and plate them in a
fresh 20% FBS containing medium in small 25 cm2 flask and incubated them at 37ºC.
Viable cell count was done before plating into the flask and calculated the percentage
survival of cells.
3.13 Animal Cell Lines and Culture Conditions
The in vitro cytotoxicity study was carried out on a range of mammalian cancer
cell lines including C6, Hep G2, MCF-7 and SK-N-SH (All from National Centre for
Cell Science, Pune, India).
3. 13.1 Hepato cellular carcinoma cell lines (HepG2)
Hep G2 cells were cultured in DMEM/F-12 (1:1) supplemented with 10% heatinactivated FBS, 200mM L-Glutamine, 2% Penicillin-Streptomycin and 2.5 µg/mL
Amphotericin-B solution (All from HiMedia Labs, Mumbai, India). Hep G2 cells
were incubated at 37°C in a humidified atmosphere of 95% air, 5% CO2 for 24-48 hrs.
Following 24-48 hrs of incubation period, the adherent cells were detached using
78
Trypsin-EDTA solution 1X/0.25% (HiMedia Labs, Mumbai, India). Cell count was
carried out using the Luna automated cell counter (Logos
(Logos Biosystems, India) based on
trypan blue dye exclusion method.
Fig. 12 Image of HepG2 cell lines
3.13.2 Human breast cancer cell lines (MCF-7)
(MCF
MCF-77 were grown in DMEM-HG
DMEM HG supplemented with 10% heat
heat-inactivated
FBS, 2% Penicillin-Streptomycin
Streptomycin and 2.5 µg/mL Amphotericin-B
B solution(All from
HiMedia Labs, Mumbai, India); and incubated at 37°C in a humidified atmosphere of
95% air, 5% CO2 for 24
24-48 hrs. Following 24-48
48 hrs of incubation period, the
adherent cells were detached using Trypsin
Trypsin-EDTA solution 1X/0.25% (HiMedia Labs,
Mumbai, India). Cell count was carried out using the Luna automated cell counter
(Logos Biosystems, India) based on trypan blue dye exclusion method.
79
Fig. 13 Image of MCF-7 cell lines
3.13.3 Human neuroblastoma cell lines (SK-N-SH)
SK-N-SH
SH cells were cultured in DMEM/F
DMEM/F-12
12 (1:1) supplemented with 10% heat
heatinactivated FBS, 200mM L-Glutamine,
L
2% Penicillin-Streptomycin
Streptomycin and 2.5 µg/mL
Amphotericin-B
B solution (All from Hi
HiMedia Labs, Mumbai, India). SK
SK-N-SH cells
were incubated at 37°C in a humidified atmosphere of 95% air, 5% CO2 for 24-48 hrs.
Following 24-48
48 hrs of incubation period, the adherent cells were detached using
Trypsin-EDTA solution 1X/0.25% (HiMedia Labs, Mumbai, India). Cell count was
carried out using the Luna automated cell counter (Logos Biosystems, India) based on
trypan blue dye exclusion method.
80
Fig. 14 Image of SK-N-SH cell lines
3.13.4. Rat glioma cell lines (C6)
C6 was maintained in DMEM
DMEM-LG supplemented with 10% heat-inactivated
inactivated feta
fetal
bovine serum (FBS), 2% Penicillin-Streptomycin
Penicillin
and 2.5 µg/mL
g/mL Amphotericin
Amphotericin-B
solution(All from HiMedia Labs, Mumbai, India); and incubated at 37°C in a
humidified atmosphere of 95% air, 5% CO2 for 24-48
48 hrs. Following 24
24-48 hrs of
incubation period, the adherent
a
cells were detached using Trypsin-EDTA
EDTA solution
1X/0.25% (HiMedia Labs, Mumbai, India).
Cell count was carried out using
the Luna automated cell counter (Logos Biosystems, India) based on trypan blue dye
exclusion method.
81
Fig. 15 Image of C6 cell lines
Fig. 16 Image of CO2 incubator (Thermo Scientifics)
3.14 In Vitro Cell Viability Assay (MTT Assay)
Cytotoxicity of the synthesised AgNPs on the cancer cells lines was determined using
MTT (3-(4,5-Dimethylthiazol
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
diphenyltetrazolium bromide) assay.
3.14.1 Under Normoxia conditions: (normal atmospheric oxygen conditions i.e,
21%)
The MTT [3-(4,
(4, 55-dimethylthiazol-2-yl)-2,5-diphenyl
diphenyl tetrazolium bromide]
assay is a test that allows the measurement
measurement of cell viability/proliferation. MTT is a
yellow dye that is converted to formazan/rezarin, an insoluble purple compound, by
the activity of intracellular mitochondrial succinatedehydrogenase enzymes. The
conversion is only able to take place in living
living cells therefore the amount of formazan
produced is directly related to the number of viable cells present. The cells are
ruptured and the formazan solubilized using a solubilization solution. The absorbance
82
of the resultant solution is read at 570nm and cell viability was calculated as a
percentage of viable cells at different test concentrations relative to the control
(untreated) cells using the following formula;
%ofcellviability =
A570 oftreatedcells
× 100
A570 ofcontrolcells
The MTT assay was performed using 200µL suspensions of each cell line which
were seeded in 96-well microplates (Corning®,
USA) at a density of 25,000
cells/well and incubated for 24hrs, after which the cells were exposed to an increasing
concentration of AgNPs (25, 50, 100, 125 and 150µg/mL) for 24 hrs. The AgNPsuntreated cells were used as controls for the experiment. All cells were seeded in
duplicates and incubated in a CO2 incubator (atmospheric with 5% CO2 and 37°C
temperature). 10 µl of 10% MTT dye (HiMedia Labs, Mumbai, India) was added to
each well and cells incubated at 37°C with 5% CO2, 95% air for 3 hrs. The cells were
viewed under a light microscope to observe if formazan had accumulated inside the
cell. The culture medium was aspirated and 100µL dimethly sulfoxide (DMSO;
Sigma-Aldrich, India) was added to each well followed by gentle pipetting to lyse the
cells. Cell viability was determined by measuring the absorbance on an ELISA
microplate reader (SPECTROstar Nano, BMG LABTECH, Germany) at 570nm.
Viability was calculated as a percentage of viable cells at different test concentrations
relative to the control (untreated) cells (% cell viability = (A570 of treated cells / A570
of control cells) ×100%). The AgNPs concentration that resulted in 50% inhibition of
cell growth was calculated as the half maximal inhibitory concentration (IC50) by
constructing
a dose-response curve.
83
Fig. 17 Spectrostar-nano-ELISA
Spectrostar
ELISA plate reader (Spectro star nano)
3.14.2 Under hypoxia conditions: (low oxygen conditions i.e, 2%)
The MTT [3-(4,
(4, 5-dimethylthiazol-2-yl)-,
5
5-diphenyl
diphenyl tetrazolium bromide]
assay is a test that allows the measurement of cell viability/proliferation. MTT is a
yellow dye that is converted to formazan/rezarin, an insoluble purple compound, by
the activity off intracellular mitochondrial succinate dehydrogenase enzymes. The
conversion is only able to take place in living cells therefore the amount of formazan
produced is directly related to the number of viable cells present. The cells are
ruptured and the formazan
mazan was solubilized using a solubilization solution. The
absorbance
ance of the resultant solution was
was read at 570nm and cell viability was
calculated as a percentage of viable cells at different test concentrations relative to the
control (untreated) cells using
u
the following formula
%ofcell
cellviability =
A570 oftreatedcells
× 100
A570 ofcontrolcells
84
The MTT assay was performed using 200µL suspensions of each cell line
which were seeded in 96-well microplates (Corning®, USA) at a density of 25,000
cells/well and incubated for 24hrs, after which the cells were exposed to an increasing
concentration of AgNPs (25, 50, 100, 125 and 150µg/mL) for 24 hrs. The AgNPsuntreated cells were used as controls for the experiment. All cells were seeded in
duplicates and incubated in a CO2 incubator (atmospheric with 5% CO2, 2% O2 and
37°C temperature). 10 µl of 10% MTT dye (HiMedia Labs, Mumbai, India) was
added to each well and cells incubated at 37°C with 5% CO2, 2% oxygen, 95% air for
3 hrs. The cells were viewed under a light microscope to observe if formazan had
accumulated inside the cell. The culture medium was aspirated and 100µL dimethly
sulfoxide (DMSO; Sigma-Aldrich, India) was added to each well followed by gentle
pipetting to lyse the cells. Cell viability was determined by measuring the absorbance
on an ELISA microplate reader (SPECTROstar Nano, BMG LABTECH, Germany) at
570nm. Viability was calculated as a percentage of viable cells at different test
concentrations relative to the control (untreated) cells (% cell viability = (A570 of
treated cells / A570 of control cells) ×100%). The AgNPs concentration that resulted in
50% inhibition of cell growth was calculated as the half maximal Inhibitory
Concentration (IC50) by constructing a dose-response curve.
3.14.3 IC50: Value Determination
A basic and simple method for calculation of the IC50 is done by linear
interpolation between the concentrations just above and just beneath 50% inhibition in
the dose response curve (= two flanking points). The IC50: Inhibitory concentration at
50% inhibition for each cell line was determined by using the spectrophotometric
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results of five different AgNPs concentrations (25, 50, 100, 125 and 150µg/mL) for
which a linear curve fit was generated. Cell viability percentages (y-axis) were plotted
against increasing concentrations of AgNPs on the x-axis. IC50 value was estimated by
using the linear equation
=
+
Where;
y = % of inhibition
x = concentration
c = constant
m = coefficient
50 is substituted for y, yielding x as the IC50 value.
(For IC50 value calculations refer appendix)
3.15 Reactive Oxygen Species (ROS) Measurement
The intracellular generation of ROS (superoxide) was measured using a cell
permeable fluorescent marker dihydroethidium (DHE). DHE upon oxidation by
superoxide anions forms a red fluorescent product (2-hydroxyethidium) within the
nucleus, intercalating the DNA (Julie Nijmeh et al. 2010). It can be detected by
fluorescent microscopy with maximum excitation and emission spectra of 518nm and
605nm respectively.
2mL cell suspension in a 6-well plate with cover slips, at a cell density of 12X105 cells per well was seeded and allowed to grow for 24h at37°C, 5% CO2. Silver
nanoparticles was added at the concentration of 75µg/ml (1ml solution) and incubated
for 24h, 37°C, 5% CO2. Following treatment, the cells were washed 1-2 times with
1mL PBS and stained the cells with 20µM DHE (1mL per well) and incubated for 3086
45 mins at 37°C, in dark. The cells were washed 1-2 times with 1mL PBS, followed
by addition of 1mL PBS. Images were captured using fluorescent microscope (EVOS
FL, Life Technologies, India) with appropriate channel settings.
3.16 Flow cytometry analysis
Flow cytometry is a technology that simultaneously measures and then
analyzes multiple physical characteristics of single particles, usually cells, as they
flow in a fluid stream through a beam of light.
In the flow cytometer, particles are carried to the laser intercept in a fluid stream. Any
suspended particle or cell from 0.2–150 micrometers in size is suitable for analysis.
Cells from solid tissue must be disaggregated before analysis. The portion of the fluid
stream where particles are located is called the sample core. When particles pass
through the laser intercept, they scatter laser light. Any fluorescent molecules present
on the particle fluoresce. The scattered and fluorescent light is collected by
appropriately positioned lenses.
A combination of beam splitters and filters
steers the scattered and fluorescent light to the appropriate detectors. The detectors
produce electronic signals proportional to the optical signals striking them. The data
are collected and stored in the computer. This data can be analyzed to provide
information about subpopulations within the sample (Givan AL, 1992).
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Fig. 18 Image of MACSQ- Flowcytometer
3.16.1 Flow Cytometric Analysis of Apoptosis and Necrosis Using Annexin V
VFITC/PI Staining
Apoptosis is a normal programmed process that occurs during the life cycle of
the cell. The process is characterized by specific morphologic changed such as loss of
plasma membrane asymmetry
asymmetry that is the earliest sign of the apoptosis and this is
usually used to detect apoptosis and necrosis in cells. In apoptotic cells, phospholipid
phosphatidylserine (PS) in the membrane is translocated from the inner to outer
plasma membrane which allows PS to bind with Annexin V which can be conjugated
to a fluorochrome and detected during apoptosis observed by microscope. While the
membrane of dead and damaged cells are permeable to propidium iodied (PI)
consequently late apoptosis in cells and necrosis in cells are detected both Annexin V
and PI. The AnnexinV-PI
PI assay allows determination of healthy cells (annexin V
V-, PI); early apoptotic cells (annexin V+, PI
PI-) and necrotic cells (annexin V -, PI +) using
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the Annexin V-FITC kit (Sigma). The kit allows detection of Annexin V bound to
apoptotic cells with fluorescein isothiocyante (FITC) which labels phosphatidylserine
sites on the membrane surface while propidium iodide (PI) labels the DNA of necrotic
cells, having permeable cell membranes.
Cells at density of 1-2X105 per well was seeded and cultured in a 6-well plate
(Corning®, USA) for 24h at 37°C in 5% CO2 humidified atmosphere and were later
exposed to 75µg/ml AgNPs for 24h. Cells treated with 5µM 5-Fluoro Uracil (5-FU;
Sigma Aldrich, India) and 200mM H2O2 were used as positive controls. After
incubation, both attached and detached cells were harvested and washed once with 1X
cold buffer and once with 1X Annexin V binding buffer (10mM HEPES, 140mM
NaCl, 2.5mM CaCl2, 0.1% FBS, pH 7.4). Cells were then suspended in 100µL
binding buffer at 1X105 cells/ml concentration and stained with 5µL Annexin V-FITC.
This was followed by an incubation period of 10-15 mins at room temperature (RT) in
dark. After incubation, the cells were washed with binding buffer and fixed with 4%
PFA (paraformaldehyde, Life Technologies, India)-0.1% Triton-X 100 (HiMedia
Labs, Mumbai, India) for 10-15 mins at room temparature.
After incubation the cells were washed with binding buffer and subjected to
0.25-0.5 mg/ml RNase (HiMedia Labs, Mumbai, India) treatment for 20mins at 37°C.
Cells were then washed and suspended in binding buffer and stained with 5µL of
10µg/mL PI and incubated for 10-15 minutes at RT. Following incubation with PI for
10-15 minutes at RT, cells were suspended in 400µL of 1X Annexin-binding buffer
and the apoptotic/necrotic populations were analysed by flow cytometer
(MACSQuant Analyser, Miltenyi Biotec, Germany).
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