CHAPTER 3

MATERIALS
&
METHODS
 Materials and Methods

3. MATERIALS AND METHODS

3.1 Cloning, expression, purification and characterization of recombinant

Survivin protein

3.1.1 Construction of Mouse Survivin (MS) gene expression vector

Survivin gene (420bp) is located on chromosome 11 in mouse. Gene encoding for

Mouse Survivin (GenBank Accession No. KU935607.1) protein was synthesized by

Geneart, USA and provided in pMAT vector codon optimized for expression in

E.coli. The gene was flanked by NcoI site on the N-terminal followed by 6 x Histidine

codons and XhoI site on the C-terminal.

pMAT:MS construct and pET28b vector were digested with NcoI and XhoI restriction

enzymes at 37°C for 2 hours. The restriction digestion mix was resolved by Agarose

gel electrophoresis (1.2%) and extracted from the gel using Gel extraction kit from

Qiagen, Germany. The gel extracted MS gene was then ligated with pET28b vector

backbone (digested with NcoI and XhoI) at 16°C for 16 hrs at a molar ratio of 1:5

(vector: insert). The ligated product was then transformed in competent E.coli DH5α

cells prepared by calcium chloride method (Sambrook et al. 1989). For

transformation, ligation mix was added to 150 µl DH5α competent cells and

incubated on ice for 20 minutes followed by heat shock treatment at 42°C for 90

seconds and immediately transferred to ice. To this transformation mix, 1 ml LB

media was added and further grown at 37°C for 1 hr. Cells were pelleted down at

8,000 rpm for 3 minutes and plated on LB agar plates containing Kanamycin

(50µg/ml) and Chloramphenicol (35µg/ml). Plates were then incubated at 37°C for

16-18 hrs. Isolated colonies obtained were grown in LB medium containing

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 Materials and Methods

Kanamycin (50µg/ml) and Chloramphenicol (35µg/ml) for 16-18hrs at 37°C and cells

were harvested by centrifugation at 8,000 rpm for 10 minutes. Plasmids were isolated

using plasmid isolation kit and digested with NcoI and XhoI to check for MS gene.

Plasmids of positive clones of DH5α were transformed in E.coli BL21(DE3)pLysS

cells. Single colony of E.coli BL21(DE3)pLysS host transformed with pET28b-MS

was grown in LB media (Himedia) containing Kanamycin and Chloramphenicol at a

final concentration of 50 µg/ml and 35µg/ml respectively for 16 hrs. The glycerol

stocks were prepared by mixing autoclaved glycerol and MS culture in the ratio of 1:3

in cryovials and stored at -70°C for long-term use.

3.1.2 Primary culture of recombinant Mouse Survivin (MS)

The 25 mL LB medium containing appropriate antibiotics was inoculated with 50µl

glycerol stock of BL21(DE3)pLysS:pET28b-MS and incubated for 16-18 hrs at 200

rpm in an orbital shaker set at 37°C.

3.1.3 Expression of recombinant MS protein

The primary culture grown overnight was used as inoculum for secondary culture for

expression of the recombinant MS protein. The primary culture was added to the fresh

medium at 1% v/v final concentration. The cells were grown in an orbital shaker at

200rpm and 37°C until the OD600 of the culture reached 0.6-0.8. At this point, the

cells were induced with 1mM IPTG for expression. The culture was harvested after 4

hours of induction and centrifuged at 6,000 X g for 10 minutes. The expression of

Survivin protein was checked on 12% SDS-PAGE.

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 Materials and Methods

3.1.4 Purification of Recombinant Survivin protein using Ni-affinity

chromatography

Harvested cells were resuspended in lysis buffer (50mM Tris-HCl, pH-7.0, 200mM

NaCl, 1mM PMSF and 5mM EDTA) and lysed by Vibra cell sonicator (Sonics &

Materials, USA) for 20 minutes at amplitude of 40% with alternating pulse On and

Off (10 seconds each). The resuspended cells were kept on ice during sonication.

Lysed cells were then centrifuged at 12,000Xg for 20 minutes at 4°C. The semi-pure

pellet of Inclusion Bodies (IBs) was washed with MQ water twice and then

resuspended in solubilization buffer (50mM Tris-HCl, pH 7.0, 8M Urea). The IBs

were allowed to solubilize for 3-4 hrs on an end-to-end rotor. Solubilized IBs were

loaded onto Ni-NTA Sepharose resin pre-equilibrated with solubilization buffer. The

protein was refolded on-column by passing a shallow gradient of decreasing urea

concentration in solubilization buffer. The bound refolded protein was eluted with

increasing concentration of Imidazole ranging from 25mM to 1,000mM.

Fractions containing the purified protein were pooled and dialyzed against 1X PBS to

remove Imidazole and Tris. Pierce Micro BCA Kit was used to determine the

concentration of the dialyzed protein.

3.1.5 Silver staining

The purification samples were subjected to SDS-PAGE and the gel was used for

silver staining. The staining was done as per the manufacturers provided protocol.

Briefly, the gel was initially fixed in the fixing solution followed by washing with

35% ethanol. Gel was then sensitized with the sensitizing solution for 2 minutes and

immediately washed thrice with distilled water (1 min X 3). Staining was done with

the staining solution for 20 minutes followed by washing with distilled water (1 min

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 Materials and Methods

X 3). The developer solution was added to the gel and kept on rocker till the bands

appeared. The reaction was stopped by washing with destaining solution.

3.1.6 Characterization of purified recombinant Survivin protein

3.1.6.1 Immunoblot analysis

Purified Survivin protein containing fractions were mixed with Laemmle buffer and

run on 12% SDS-PAGE. The bands obtained were transferred to nitrocellulose

membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, USA). The blotted

membrane was blocked with 2% BSA and 1% skimmed milk in PBS for 1 hr at 37°C.

After washing with PBS, the membrane was incubated with anti-Survivin monoclonal

antibody (Santa Cruz Biotechnology) diluted 1:1,000 in PBS for 1 hr at RT. The

membrane was then washed with PBS thrice and incubated with horse radish

peroxidase (HRP)-conjugated anti-mouse goat antibody (IgG) (Jackson

Immunoresearch) for 1hr at RT. After washing, the membrane was developed with

1mg/ml 3,3’-diaminobenzidine (DAB) (Sigma), 1µl/ml Hydrogen Peroxide (H2O2)

dissolved in PBS for 10-15 minutes at RT. After the bands developed, the membrane

was washed with Milli-Q water.

3.1.6.2 Analysis of secondary structure using Circular Dichroism (CD) spectroscopy

A far-UV CD spectrum was recorded using J810 spectropolarimeter (Jasco, Japan).

The spectrum was acquired in the wavelength range of 200-250 nm at 25°C to study

the secondary structure of the purified protein (200µg/ml in PBS).

3.1.6.3 Tertiary structure analysis using Fluorescence emission spectroscopy

The fluorescence spectra for purified protein were recorded using Cary Eclipse

spectrofluorimeter (Varian, USA). Purified protein at concentration of 50µg/ml in

PBS was added to the Quartz cuvette. Samples were excited at 280nm and emission

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 Materials and Methods

spectra were recorded in the wavelength range of 300 – 400nm. Fluorescence spectra

represented in the results section is the average of three spectra recorded.

3.1.6.4 Limited Trypsin proteolysis

Purified recombinant Survivin protein was subjected to trypsin proteolysis. Survivin

has a lysine residue at position 115 that is exposed when properly folded. Digestion of

Survivin with trypsin produces two fragments, one of which is approximately 15kDa.

To confirm that the protein was properly folded, purified protein was taken in a

microcentrifuge tube and incubated with 1 µl of 100 ng/ml solution of Trypsin. 20µl

samples were removed from the reaction mixture after 1, 2, 5 and 10 minutes. The

reaction was stopped by addition of 5µl of 1mM PMSF (prepared in ethanol). The

samples retrieved at different time points were run on SDS-PAGE along with an

undigested sample.

Estimation of purified protein

The concentration of protein was estimated by BCA (Thermo Scientific, USA). The

standard curve was prepared by serially diluted BSA solution provided in the kit.

Samples were diluted to appropriate concentration and 100µL of each was added to

the plate. The assay reagent was prepared as per the manufacturer’s protocol and

100µL was added to standard and sample wells and incubated at 37°C for 30 minutes.

The samples were cooled down to room temperature before recording the absorbance.

The absorbance was read at 540 nm using ELISA reader.

3.1.6.5 Confirmation of immunogenicity of Survivin in rabbit

To generate polyclonal anti-sera against Survivin, the New Zealand white rabbit was

immunized (Day 0) with 100µg purified recombinant Survivin protein adsorbed on

Alhydrogel (Brenntag, Denmark) combined with 5 X 108 cells of heat-killed

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 Materials and Methods

Mycobacterium indicus pranii. Two booster doses of the same combination were

administered at Day 15 and Day 30. The serum was collected every 15 days

beginning from pre-immunization bleed on Day -1 followed by day 15, 30, 45, 60 and

75.

The antibody titers against Survivin were determined by ELISA. The plates were

coated with 100µl of recombinant Survivin (2µg/ml) and incubated overnight at 4°C.

After washing with 0.05% PBST, plates were blocked with 2% BSA at 37°C for 2

hrs. Plates were then washed with PBST thrice and incubated with 100µl of 1:1,000

and 1:2,000 dilutions of sera in 1X PBS. The plates were incubated at 37°C for 1 hr.

Following washing with PBST, the plates were then incubated with 100 µl of anti-

rabbit IgG antibody conjugated with horseradish peroxidase (HRP) at a dilution of

1:10,000 for 1 hr at 37°C. After PBST washing, 100µl of 1mg/ml solution of O-

phenylenediamine (OPD) prepared in Citrate buffer was added to the wells and the

reaction was stopped with 50µl 2N H2SO4. The optical density was recorded at

490nm using ELISA reader (ELX 800MS, Biotek, USA).

3.1.7. in vitro culture of 4T1 cells

Murine breast cancer cell line, 4T1 (CRL 2539) was obtained from American Type

Cell Culture Collection, USA. The cells were maintained in high glucose Dulbecco’s

Modified Eagle’s medium (DMEM) (with L-glutamine (4mM), glucose (4.5g/L) and

sodium bicarbonate (3.7g/L) in 25mM HEPES buffer with sodium pyruvate)

supplemented with 10% FBS, 100 units/mL Penicillin and 100 µg/ml Streptomycin at

37°C in CO2 incubator (N-Biotek, Korea) with a humidified atmosphere. Cells were

cultured in tissue culture flasks till 50% confluence and passaged by treating with

Trypsin-EDTA (0.25% Trypsin, 0.02% EDTA) solution. Single cell suspension of

34
 Materials and Methods

4T1 was prepared in sterile 1X PBS, counted using cell counter and used for

developing tumor in mice.

3.1.8 Screening of 4T1 cells for Survivin expression

3.1.8.1 Flow cytometry

4T1 cells were cultured in DMEM and trypsinized at 50-60% confluence. Single cell

suspension was prepared in 1X PBS and counted using Neubauer’s chamber. 1 X 106

cells were taken in each FACS tube and fixed with 4% Formaldehyde for 10 minutes

on ice. Cells were immediately washed with FACS buffer (2% BSA in 1X PBS) and

incubated with 1:20 dilution of monoclonal anti-Survivin antibody (Santa Cruz

Biotechnology) overnight at 4°C. After washing with FACS buffer thrice, FITC-

conjugated anti-mouse antibody was added to respective tubes and incubated at room

temperature for 1 hr. Data was acquired on FACS Verse after washing with FACS

buffer.

3.1.8.2 Cell ELISA

The surface expression of Survivin was assessed on adhered 4T1 cells. 5,000 cells

(100µl/well) were seeded in a 96-well tissue culture plate and incubated at 37°C in a

CO2 incubator overnight. The media was aspirated from all wells and after washing

with 1 X PBS the cells were fixed with 4% formaldehyde for 10 minutes. The

blocking solution was added to the wells to prevent non-specific binding of the

antibody. For intracellular expression, one set of wells were permeabilized with

0.01% Triton-X 100 for 20 minutes. After washing, anti-Survivin antibody was added

in serial dilution starting from 1µg/100µl till 0.0625µg/100µl. Antibody was added in

duplicates for each concentration. The plate was incubated at 37°C for 1 hr followed

by washing with 1X PBS thrice without disturbing the cells. The anti-mouse HRP-

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 Materials and Methods

conjugated antibody was added to the wells and incubated for 1 hr at 37°C. 100µl

OPD was added to each well and allowed to react for 15 minutes. The reaction was

stopped by adding 50µl 2N H2SO4. The absorbance was read at 490nm.

3.1.9 in vitro cell cytotoxicity assay using monoclonal antibody against Survivin

The in vitro cytotoxicity of anti-Survivin antibody on 4T1 cells was assessed by MTT

assay.

10,000 4T1 cells were seeded per well in a 96 well plate. Plate was incubated

overnight at 37°C in a CO2 incubator.

The media was removed from all wells and monoclonal anti-Survivin antibody was

added in serial dilution starting from 20µg in duplicates. The dilution was done in

PBS and the media volume was kept equal in all wells (100µl DMEM +100µl

antibody in 1X PBS). Complete DMEM along with PBS was taken as control.

The cells were incubated with antibody overnight in a CO2 incubator.

Next day, the antibody was aspirated from the wells and 100µl rabbit complement

(12% in complete media) was added to the wells. The cells were incubated for 2 hours

in the CO2 incubator. In 2 wells of control, only Complement (diluted in DMEM) was

added.

The complement was removed and 50µl of MTT reagent (5mg/ml) was added to all

the wells and incubated at 37°C for 2 hours (till formazan crystals were formed)

25µl DMSO was added to each well to solubilize crystals and the plate was read at

540nm.

Cytotoxicity was calculated as follows:

% Cytotoxicity= (C-T) X 100

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 Materials and Methods

C- Control O.D.

T- Test O.D.

Animal ethics protocol

The in vivo anti tumor studies were conducted in 6-8 weeks old female Balb/c mice

(inbred). Pathogen-free mice were procured from National Institute of Nutrition,

Hyderabad. Animals were maintained at animal facility of Amity University, Uttar

Pradesh with proper access to food and water. All the procedures were strictly in

accordance with the guidelines of Committee for the Purpose of Control and

Supervision of Experiments on Animal guidelines (CPCSEA) for care and use in

laboratories. Institutional Animal Ethics Committee (IAEC) of Amity University

Uttar Pradesh approved all the experimental protocols.

3.2 in vivo tumor preventive studies with recombinant Survivin supplemented

with heat-killed Mycobacterium indicus pranii (MIP)

6-8 weeks old female Balb/c mice were randomly divided into different groups. The

studies were conducted in 2 parts. Mice were divided into different groups consisting

of 6 mice/group. The control or untreated group was immunized with PBS and the test

groups were administered 5µg, 10µg, 20µg and 50µg of recombinant Survivin

adsorbed on alum along with 5 X 106 cells of heat killed MIP per mice

intramuscularly in each group (irrespective of the dose of antigen). Two booster doses

fortnightly after the primary immunization were given to mice in each group. 60 days

post last immunization; all the mice were challenged with 3 X 105 cells of 4T1 in

100µl PBS (prepared as described earlier) subcutaneously above the right flank. Day

of tumor challenge was denoted as Day 0 and the days of immunizations were labeled

as Day -90, -75 and -60. Tumors developed in mice and were palpable after 12-15

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 Materials and Methods

days post tumor inoculation. The third booster was given to the mice at the same day.

Serum was collected fortnightly since first immunization till the end of the study. Sera

collected were analyzed for total IgG titer and anti-Survivin isotype specific

antibodies.

Tumor volume was calculated, after measuring the tumor size bi-dimensionally using

the digital Vernier calipers, by the formula:

𝑎 𝑋 𝑏!
2

where,

‘a’ is the largest diameter, and

‘b’ is the perpendicular diameter

The tumor volume was calculated till Day 28 after tumor inoculation. Tumor

inhibition in the test groups was calculated with respect to the tumor volume in

untreated group of mice using the following formula

𝑇𝑉!"#$%"& !"# ! − 𝑇𝑉!"# ! − (𝑇𝑉!"#$ !"# ! − 𝑇𝑉!"#$ !"# ! )

𝑋 1000
(𝑇𝑉!"#$%"& !"# ! − 𝑇𝑉!"# ! )

where,

𝑇𝑉!"#$ !"# ! = Median tumor volume of treated group at Day X,

𝑇𝑉!"#$ !"# ! = Median tumor volume of treated group at Day 0

𝑇𝑉!"#$%"& !"# ! = Median tumor volume of control group at Day X

𝑇𝑉!"# ! = Median tumor volume of control group at Day 0.

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 Materials and Methods

3.3 Therapeutic study to check tumor inhibitory potential of Survivin-MIP

combination

The potential of Survivin-MIP combination as an anti-tumor therapy was studied by

challenging all mice (6-8 weeks old) with 1 X 105 cells of 4T1 subcutaneously in the

right flank (Day 0). Mice were then randomly divided into 4 groups: 1) Untreated, 2)

Survivin (10µg)-MIP, 3) Survivin (20µg)-MIP and 4) Tamoxifen (5µg). First

immunization was done on Day1. Purified Survivin was adsorbed on alhydrogel

before immunization. The Survivin-MIP duo was given intramuscularly and

Tamoxifen was administered subcutaneously above left flank. Tamoxifen was

prepared as a stock of 1mg/ml in DMSO and 5µl of this was added to 95µl PBS for

immunizing mice. The untreated group was given 100µl of 1X PBS. Three booster

doses were given for group 2 and 3 at Day 8, Day 15 and Day 23. 15 doses of

Tamoxifen were given every alternate day. The tumor size was measured regularly

and tumor volume was calculated as described previously. Sera for each group was

collected at 15 days interval till the end of the study to check for total anti-Survivin

IgG and levels of IgG subclasses induced in different immunization groups.

3.4 Evaluating anti-tumor effects of Survivin-MIP and LHRH-MIP alone and in

combination

The study protocol for the comparative study of Survivin-MIP and LHRH-MIP was

similar to the Survivin-MIP alone study at different doses (described above). Briefly,

in this study, both the recombinant proteins were administered at the dose of 20µg

along with MIP (5 X 106 cells) individually as well as in combination i.e.

Survivin+LHRH+MIP. The tumor cells were inoculated one week after last

immunization and the tumor volume was calculated till Day 28 post tumor inoculation

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 Materials and Methods

as per the formula given above. Secondary tumor development was studied in each

group by counting the number of metastatic nodules in the lungs.

3.5 Indirect ELISA for total anti-Survivin IgG titers as indicator of Humoral

response

Sera samples from all the in vivo studies were tested for anti-Survivin antibody titers

by indirect ELISA. High-binding 96-well flat-bottom ELISA plates were coated with

100µl of 5µg/ml solution of purified recombinant Survivin prepared in carbonate

buffer and incubated at 4°C overnight. Plates were washed with phosphate buffered

saline supplemented with 0.05% Tween-20 (PBS-T) thrice and then blocked with

250µl of 5% BSA (Bovine Serum Albumin) in PBS for 1 hour at 37°C. After washing

with PBS-T thrice, 100µl of serum samples (1:5,000) were added in duplicates and

incubated for 2 hours at 37°C. Following washing with PBS-T, secondary goat anti-

mouse antibody conjugated with HRP was added and incubated at 37°C for 1 hour.

Plates were then washed extensively for 4 times. The reaction was initiated by

addition of 100µl of ortho-phenylenediamine (OPD) dissolved in citrate-phosphate

buffer at the final concentration of 1mg/ml along with H2O2 to each well. The reaction

was carried out for 15 minutes in dark and then stopped by adding 50µl/well of 2N

H2SO4. The absorbance was recorded at 490 nm using an ELISA reader.

3.6 Detection of levels of Survivin specific IgG subclasses in the sera

Briefly, the 96-well microtitre plates were coated with 2µg/ml of recombinant

Survivin and incubated overnight at 4°C. The unbound sites were blocked with 2%

BSA after washing with PBS-T and incubated at 37°C for 1 hr. Followed by washing,

the serum samples were added at a dilution of 1:500 and Survivin-specific antibodies

in the serum were allowed to bind with the antigen at 37°C for 1 hr. Post washing, the

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 Materials and Methods

HRP-labeled IgG subtype (IgG1, IgG2a and IgG2b) specific antibodies were added to

the wells to detect their levels in different immunization groups. After washing, plates

were incubated with 100µl of OPD solution containing H2O2 for 15 minutes at 37°C

and the reaction was stopped with 50µl 2N H2SO4 and absorbance was measured by

ELISA reader at 450nm.

The ratio of IgG1 and IgG2a was calculated to determine the Th1/Th2 biased

response.

3.7 Immunological responses induced against alum adjuvanted Survivin and

MIP in mice

To study the immunological responses induced by immunization of alum adsorbed

Survivin (either with MIP or without MIP), 6-8 weeks old Balb/c mice were randomly

divided into 5 groups: Group-1 was given PBS (Control), Group-2 was immunized

with 20µg Survivin alone, Group-3 received 20µg Survivin (+MIP), Group-4 was

administered with 50µg Survivin and Group-5 was immunized with 50µg Survivin

(+MIP). Each group of mice received two booster immunizations at 15 days interval

post primary immunization. All immunizations were given intramuscularly. 15 days

after the last immunization (Day 45) mice were sacrificed and their spleen was

removed to study the antigen-specific immune response.

3.7.1 Preparation of splenocytes

The spleen was removed aseptically and washed with RPMI in sterile conditions. It

was then crushed with the plunger of 10mL syringe to obtain a homogenous cell

suspension. The cell suspension was then passed through a cell strainer to get a single

cell suspension of splenocytes. The RBCs were lysed by resuspending the cells in

RBC lysis buffer (150mM Ammonium Chloride, 12mM Sodium bicarbonate and

41
 Materials and Methods

0.1mM EDTA). The suspension was centrifuged after 10 minutes at 400g for 5

minutes. The pellet was washed with complete RPMI media and used for further

assays.

3.7.2 Splenocyte Proliferation assay

To study recall response against Survivin in the splenocytes of Survivin-immunized

mice, 5 X 105 cells (100µl) of each mice were seeded in 96-well tissue culture plate

and stimulated with 100µl of 0µg, 5µg, 10µg, 20µg and 50µg/ml recombinant

Survivin (prepared in 10% RPMI) for 120 hours at 37°C. PMA(50ng)/ Ionomycin

(300ng) was taken as positive control. After 120 hrs, the cell suspension was removed

and centrifuged at 1500 rpm for 5 min. Splenocytes were collected in separate tubes

and washed with PBS twice. For dead cell staining, the cells were incubated with

Fixable violet dye (diluted in 0.9% NaCl) for 45 minutes in dark. After washing with

PBS, cells were permeabilized with BD Perm buffer. To stain the proliferating cells,

Ki-67 antibody was added and incubated at 4°C overnight in dark. After washing

twice with PBS, data was acquired on FACS Verse. The percentage of live,

proliferating cells (V450-ve, Ki-67+ve) was determined for each animal. The average

for the group was calculated.

3.7.3 Detection of levels of cytokines in the supernatant

Cell culture supernatants collected from the in vitro proliferation assay after 24 hrs

were assayed for determining cytokine levels using e-biosciences ELISA kits. Briefly,

for this assay the splenocytes (5 X 105 cells) from each mouse were cultured in

complete RPMI for 24hrs along with different concentrations (0µg, 5µg, 10µg, 20µg

and 50µg/ml) of recombinant Survivin antigen. The supernatant were harvested for

each group in separate tubes and stored at -70°C till the assay was performed.

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 Materials and Methods

Cytokine levels were measured as per the protocol suggested by the manufacturer.

Briefly, 96-well microtitre plates were coated with 100µl/well recombinant anti-

mouse capture antibodies (anti- IFN-gamma, anti-IL-2 and anti IL-4) at the

recommended dilutions and incubated at 4°C overnight. The plates were then washed

and blocked using blocking buffer (200µl/well) at 37°C for 2 hrs. Standards were

added in serial dilution (IFN-gamma: 30-2000pg/ml; IL-2: 3-200pg/ml and IL-4: 8-

500pg/ml) and the test samples were added in duplicates at appropriate dilution. The

plate was then incubated for 2 hrs at 37°C. The dilutions were prepared in blocking

buffer. After washing, detection antibody (100µl/well) was added to the respective

wells and incubated for 1 hr at 37°C. Subsequently, Avidin-HRP enzyme was added

to each well for 30 minutes. After extensive washing, TMB substrate was added and

after incubation of 15 min at room temperature, reaction was stopped using 2N H2SO4

and absorbance was read at 450nm.

3.7.4 Surface staining of splenocytes

The splenocytes from each mouse were checked for expression of different CD

markers and different T cell populations induced upon treatment with different

combinations of Survivin and adjuvants. The splenocytes were subjected to flow

Cytometry for analysis of cellular immune response. Different panels of antibodies

were prepared for detection of different T cell populations. The splenocytes (0.5X105

cells) were first washed with FACS buffer (2% BSA in 1X PBS) and then incubated

with Fc block antibody to prevent non-specific binding of the antibodies for 30

minutes at 4°C. The cells were then fixed with 4% formaldehyde for 10 minutes at

4°C followed by washing with FACS buffer. The splenocytes were then stained for

different panels of T cell marker antibodies.

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 Materials and Methods

For staining, fluorochrome-conjugated mouse monoclonal antibodies specific for

CD3, CD4, CD8, CD69, CD25 and FoxP3 were used. Samples were run on

FACSVerse and data was analysed using FlowJo software. For Treg population,

splenocytes stained with CD4 and CD25 were permeabilized with BD Perm buffer

and then allowed to stain with anti-FoxP3 antibody.

3.7.5 Statistical Analysis

Statistical analysis of the datasets for tumor volume was done using two-tailed

Student’s t-test. The statistical significance of the immune response generated by

different immunization groups was determined using Tukey’s Multiple Comparison

Test. All the data were considered significant at p<0.05. The analysis was done using

Graph Pad Prism software.

44