III.

MATERIALS AND METHODS

Cleaning of glassware

The glassware used were primarily soaked overnight in a cleaning solution and
washed thoroughly under running tap water. Then they were rinsed with a detergent solution
followed by a thorough wash with tap water and a final rinse with distilled water and dried.

Preparation of cleaning solution

Potassium dichromate - 60 g
Conc. H2SO4 - 60 mL
Distilled water - 1000 mL

The cleaning solution was prepared by adding potassium dichromate in warm water,
dissolved, cooled and slowly added with sulphuric acid. It was thoroughly mixed and used for
cleaning of glassware (Schmerber et al., 2005).

Sterilization

The washed, dried glassware and the media were sterilized in an autoclave at 121oC at
15 psi for 20 min.

Chemical and Reagents

Chemicals used in the investigation were analytical grade, obtained from the Sisco
Research Laboratories Pvt. Ltd., Mumbai; Himedia Laboratories Pvt. Ltd., Mumbai; Ranbaxy
Fine Chemical Pvt. Ltd., New Delhi; Thomas Baker (Chemicals) Pvt. Ltd., Mumbai and
Qualigens Fine Chemical, Mumbai. Authentic standard and fine chemicals such as
astaxanthin, Dulbecco’s Modified Eagle Medium (DMEM), 0.25% Trypsin-EDTA solution,
sodium bicarbonate solution, bovine serum albumin (BSA), low melting agarose, MTT,
propidium iodide, ethidium bromide, acridine orange and anti-β-actin were purchased from
the Sigma Chemicals Co., (St. Louis, USA). Antibodies were purchased from Sigma

54
chemicals Co., (St. Louis, MO, USA). Primary antibodies against Bcl-2, Bax, PCNA,
Caspase and β-actin, and the respective HRP-labelled secondary antibodies were purchased
from the Santa Cruz Biotechnology and Genei, Bangalore, India.

Haematococcus lacustris

In the present attempt, Haematococcus lacustris (Girod-Chantrans) Rostafinski

(= Haematococcus pluvialis Flotow) HPI- 001 was obtained from the Algal Culture
Collection, Centre for Advanced Studies in Botany, University of Madras. This strain was
isolated from the Himachal Pradesh, India, and an another one strain, Haematococcus
lacustris SAG-19a was obtained from the Gottingen Culture Collection, Germany. The latter
isolate was treated as Control / Reference alga in the present attempt. Both strains were
maintained in the Modified 3N-BBM+V medium at 25 ± 1oC, under 30μEm-2 s-1
light irradiance and photoperiod of 12/12 (Light/Dark). The cultures were thoroughly mixed
manually twice a day and investigated under different laboratory conditions. Growth study
was conducted on both strains for a period of 30 days. At every 5 days interval the algae
samples were estimated for total protein (Brad Ford, 1976), total carbohydrates (Dubois et
al., 1956), total lipids (Folch et al., 1957) and photosynthetic pigments such as Chlorophyll
a, Chlorophyll b and total carotenoids (Lichtenthaler, 1987) and astaxanthin (Davies, 1976).
In addition cell numbers were also recorded using Haemocytometer. Axenic culture of the
algae was used in the following experiments.

Maintenance of stock culture

The two strains of Haematococcus lacustris was maintained on agar and in liquid
Modified 3N- BBM+V medium.

55
Composition of Modified Bold-Basal Medium with 3-fold nitrogen and vitamins
(3N-BBM+V) (Kanz and Bold, 1969; www.ccap.ac.uk)

Macroelements
Stock 1. NaNO3 - 25.0 g/L
Stock 2. MgSO4.7H2O - 7.5 g/L
Stock 3. NaCl - 2.5 g/L
Stock 4. K2HPO4.3H2O - 7.5 g/L
Stock 5. KH2PO4 - 17.5 g/L
Stock 6. CaCl2.2H2O - 2.5 g/L
Stock 7. Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2Mo4.2H2O - 4.0 mg
Na2EDTA - 0.75g
Glass distilled water - 1000 mL
pH - 7.5

Stock 8. Vitamin B1
Thiamin hydrochloride of 0.12 g was glass dissolved in 100 mL distilled water and
filters sterilized.

Stock 9. Vitamin B12

Cyanocobalamin of 0.1g was dissolved in 100 mL glass distilled water. One mL of this
solution was made up to 100 mL with glass distilled water and filter sterilized.

Preparation of medium
Stock : 1 - 30.0 mL
Stocks: 2 to 6 each - 10.0 mL
Stock: 7 - 6.0 mL
Stocks: 8 and 9 each - 1.0 mL

56
 Added the stocks 1-7 as described above and made up to 1 liter with deionized water
and sterilized. To this, the stocks of 8 and 9 were added aseptically.

The agar slants and liquid culture of H. lacustris were subcultured at every 4 and 2
weeks intervals, respectively, under culture conditions.

Elimination of contaminants

Cyanobacteria

The cyanobacterial contaminants in the sample were eliminated by treatment with

3000 ppm of Streptomycin sulphate for 30 minutes under 30 µEm-2s-1 light intensity and then
transfering to an antibiotic-free basal medium (Rengasamy et al., 1987).

Growth study:

The culture flasks containing 270 ml of basal medium was inoculated with 30 ml of
optimally grown algae culture of H. lacustris and kept under the laboratory condition.
The growth study was conducted for a period of 30 days. At every 5 days interval, the
amounts of total protein, carbohydrate, lipid and photosynthetic pigments such as
Chlorophyll a, b and total carotenoids and astaxanthin were estimated and recorded.
In addition, the number of cells/mL was also recorded using Haemocytometer.

Cell count

The cell count was measured using a ‘Neubauer’ Haemocytometer and the mean of
the cell numbers recorded in four chambers was calculated and expressed as 10 4 cells/mL.

Growth curve

The cell counts expressed as 104 cells/mL were plotted against the respective days to
obtain growth curves.

Specific Growth rate, Division per day, Generation time

From the cell number data, the specific growth rate, division per day, and generation
time were calculated using the following formula (Levasseur et al., 1993).

57
 Specific growth rate; K’ = Ln (N2/N1) / (t2-t1)

Division per day; Div.day-1 = K’/Ln2

Generation time; Gen’t = 1/ Div.day-1

Where N1 and N2 = Cell number at time 1 (t1) and time 2 (t2) respectively.

Determination of pigments (Lichtenthaler, 1987)

Extraction of pigments

The different photosynthetic pigments such as Chl a, Chl b, and total carotenoids of
Haematococcus sample were extracted by following methods of Lichtenthaler (1987).
Five ml of culture sample was centrifuged (R-8C; Remi Instruments Ltd, Mumbai, India) at
5000 rpm for 10 min and the supernatant discarded. The algal pellet was added with 5 mL of
100% acetone and macerated using pestle and mortar. Then the sample was centrifuged at
5000 rpm for 10 min after an overnight incubation at 4°C wrapped in black paper. The
supernatant was collected and the absorbance was recorded at 661.6 nm, 644.8 nm, 470 nm,
and 490 nm wavelengths in Hitachi U2900 UV-Vis spectrophotometer (Hitachi, Japan) in
standard quartz cuvette (190 – 2500 nm), path length 10 mm. The astaxanthin content in the
acetone extract was estimated spectrophotometrically measured at 490 nm. The per unit
volume of astaxanthin content was calculated by using the method of Davies, (1976).

Estimation of pigments
The pigments were quantified by using the following formula:

Chlorophyll a (mg/L) = 11.24 × A661.6 - 2.404 × A644.8

Chlorophyll b (mg/L) = 20.13 × A644.8 - 4.19 × A661.6

Total carotenoids 1000 × A470 - 1.9 × Chl a - 63.14 × Chl b

=
(mg/L) 214

A480 × Volume of sample × dilution factor × 10

Astaxanthin (mg/L) =
2500

A = Absorbance measurement

58
 All the experiments were carried out in triplicates. The mean values are presented in
the thesis.

Extraction and Estimation of Total protein (Bradford, 1976)

Reagents

0.1 M Phosphate buffer (pH 7.0)

Sixty-one ml of 0.2 M Na2HPO4 was added with 39 ml of 0.2 M NaHPO4 and made up
to 200 ml with glass distilled water.

Coomassive Brilliant Blue reagent

One hundred mg of CBB G-250 was dissolved in 50 ml of 93% ethanol. To this 100 ml
of 85% phosphoric acid was added and diluted to 1000 ml with glass distilled water.

Extraction of protein

Five ml of culture was centrifuged at 5000 rpm for 10 min. The pellet was
homogenized with 5 ml of Sodium phosphate buffer at pH 7.0 was sonicated and then
centrifuged at 5000 rpm for 10 min. The supernatant was used for estimating total protein.

Estimation of Protein

To 0.5 ml of sample 5.0 ml of protein reagent was added and mixed thoroughly. The
absorbance was measured at 595 nm against a blank. The amount of protein was calculated
by using a standard graph prepared with Bovine Serum Albumin ranging from 10 to 100 g
/mL.

Extraction and Estimation of Total carbohydrate (Dubois et al., 1956)

Reagents:

Phosphate buffer 0.1M (pH 6.8)

Sodium phosphate buffer (0.1M, pH 6.8) was prepared by mixing 49 ml of 0.2 N

Na2HPO4 and 51 ml of 0.2 N NaH2PO4 and made up to 100 ml with glass distilled water.

59
5% Phenol

Five gram of phenol crystals was dissolved in 100 ml of glass distilled water and
refrigerated.

Sulphuric acid

The sulfuric acid (96%) used was of analytical grade.

Extraction of Total carbohydrates

Five ml of culture was taken and centrifuged at 5000 rpm for 10 min. The pellet was
homogenized with 5 ml of Sodium phosphate buffer at pH 6.8 in a Sonicator
(UP2005 Ultrasound Technology) and centrifuged at 5000 rpm for 10 min. The supernatant
was collected for total carbohydrate estimation.

Estimation of Total carbohydrate

One ml of 5% phenol and 5 ml of H2SO4 were added to 1 ml of sample, and the

mixture was thoroughly vortexed. The optical density of the mixture was measured at 490
nm after allowed to stand at room temperature for 30 min. The different concentrations of D-
glucose ranging from 10-100 µg/mL were used as the standard.

Extraction and Estimation of total lipid (Folch et al., 1956)

Reagents

Chloroform: Methanol (2:1v/v)

Fifty ml of Chloroform was mixed with 25 ml of Methanol.

Physiological saline (0.9%)

Sodium chloride of 900 mg was dissolved in glass distilled water and made up to 100
ml.

60
Phosphovanillin reagents

Eighty ml orthophosphoric acid and 20 ml of glass distilled water were mixed and 200
mg of vanillin was added to the mixture.

Extraction of Total lipid

Five ml of culture was centrifuged at 5000 rpm for 10 min and the resultant pellet was
homogenized with pestle and mortar with 6.0 ml of Chloroform: Methanol (2:1).
It was then transferred to a separating funnel and added with 2.0 ml of 0.9 ml NaCl solution
and mixed well. This mixture was left undisturbed for overnight. The lower chloroform phase
containing lipid 0.5 ml was collected in a vial and allowed the solvent to evaporate at room
temperature. After evaporation, the pellet was collected and stored for further studies.

Estimation of Total lipid

The tubes containing the pellet were added with 0.5 ml of conc. H2SO4, mixed well
and closed with glass marbles. They were kept in a boiling water bath for 10 min and allowed
to cool at room temperature. To 0.2 ml of this mixture, 5.0 ml of phosphovanillin reagent was
added, mixed well and allowed to stand for 30 min. The colour developed was read at 520
nm. The standard graph was prepared by using cholesterol ranging from
10-100 µg/mL.
Effect of Different Media

The two strains of H. lacustris such as H. lacustris HPI- 001 and H. lacustris SAG-
19a were inoculated grown in different media such as BBM, 3N-BBM+V, BG – 11, and
Modified CHU – 13 and recorded in different parameters.

61
Composition of Bold-Basal Medium (Kanz and Bold, 1969)

Macroelements
Stock 1. NaNO3 - 25.0 g/L
Stock 2. MgSO4.7H2O - 7.5 g/L
Stock 3. NaCl - 2.5 g/L
Stock 4. K2HPO4.3H2O - 7.5 g/L
Stock 5. KH2PO4 - 17.5 g/L
Stock 6. CaCl2.2H2O - 2.5 g/L
Stock 7. Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2Mo4.2H2O - 4.0 mg
Na2EDTA - 0.75g
Glass distilled water - 1000 mL
pH - 7.0

Stock 8. Vitamin B1

Thiamin hydrochloride of 0.12g was glass dissolved in 100 mL distilled water

and filters sterilized.

Stock 9. Vitamin B12

Cyanocobalamin 0.1 g was dissolved in 100 mL glass distilled water. One mL of this
solution was made up to 100 mL with glass distilled water and filter sterilized.

Preparation of medium
Stock: 1 - 10.0 mL
Stocks: 2 to 6 each - 10.0 mL
Stock: 7 - 6.0 mL
Stocks: 8 and 9 each - 1.0 mL

62
Added the stocks 1-7 as described above and made up to 1 liter with deionized water and
sterilized. To this, the stocks of 8 and 9 were added aseptically.

Composition of Modified Bold-Basal Medium with 3-fold nitrogen and vitamins

(3N-BBM+V) (Kanz and Bold, 1969)

Macroelements
Stock 1. NaNO3 - 25.0 g/L
Stock 2. MgSO4.7H2O - 7.5 g/L
Stock 3. NaCl - 2.5 g/L
Stock 4. K2HPO4.3H2O - 7.5 g/L
Stock 5. KH2PO4 - 17.5 g/L
Stock 6. CaCl2.2H2O - 2.5 g/L
Stock 7. Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2Mo4.2H2O - 4.0 mg
Na2EDTA - 0.75g
Glass distilled water - 1000 mL
pH - 7.5

Stock 8. Vitamin B1

Thiamin hydrochloride of 0.12g was glass dissolved in 100 mL distilled water

and filters sterilized.

Stock 9. Vitamin B12

Cyanocobalamin 0.1g was dissolved in 100 mL glass distilled water.

One mL of this solution was made up to 100 mL with glass distilled water and filter
sterilized.

63
Preparation of medium
Stock : 1 - 30.0 mL
Stocks: 2 to 6 each - 10.0 mL
Stock: 7 - 6.0 mL
Stocks: 8 and 9 each - 1.0 mL

Added the stocks 1-7 as described above and made up to 1 litre with deionized water and
sterilized. To this, the stocks of 8 and 9 were added aseptically.

Composition of BG- 11 Medium (Rippka et al., 1979)

NaNO3 - 1500 mg
K2HPO4.3H2O - 40 mg
MgSO4.7H2O - 75 mg
CaCl2.2H2O - 36 mg
Citric acid - 6 mg
Ferric Ammonium citrate - 6 mg
EDTA (Na2 Mg Salt) - 10 mg
Na2 CO3 - 20 mg
Microelements
H3 BO3 - 289 mg
MnCl2.4H2O - 109 mg
ZnCl2.7H2O - 22 mg
Na2Mo4.2H2O - 39 mg
CuSO4.5H2O - 79 mg
Co(NO3)2.6H2O - 50 mg
Glass distilled water - 1000 mL
pH - 7.5

64
Composition of Modified CHU – 13 medium (Yamaguchi et al., 1987)

Macroelements

KNO3 - 371 mg
K2HPO4 - 80 mg
MgSO4.2H2O - 200 mg
CaCl2.2H2O - 107 mg
Citric acid - 20 mg
Ferric Citrate - 6 mg
Citric acid - 100 m
Microelements - 1 mL
Glass distilled water - 1000 mL
pH - 7.5

Microelements: (Stock 100 mL glass distilled water)

H3 BO3 - 289 mg
MnCl2.4H2O - 181 mg
ZnSO4.7H2O - 22 mg
Na2Mo4.2H2O - 39 mg
CuSO4.5H2O - 8 mg
Co(NO3)2.6H2O - 5 mg
H2SO4 - 1 mL
pH - 7.5

Effect of different pH

The two algal strains were inoculated in media with different pH viz, 6.0, 6.5, 7.0
(control) 7.5, 7.8 and 8.0 respectively, adjusted with 1 N NaOH and 1 N HCl before
sterilization and the different parameters were recorded.

65
Effect of different light intensities

The two algae were kept under different light intensities such as 10, 20, 30 (control)
and 40 µEm-2 s-1 and the results were recorded.

Effect of different concentrations of Sodium nitrate

The basal medium devoid of NaNO3 was supplemented with different concentrations
of Sodium nitrate such as 3.0, 6.0, 9.0 (control), 12.0, and 15.0 mM and recorded different
parameters of the two test algae.

Effect of different concentrations of Dipotassium phosphate

The different concentrations of dipotassium phosphate such as 0.15, 0.30, 0.45

(control), 0.60 and 0.75 mM was added to the basal medium devoid of K2HPO4.3H2O and
different parameters were recorded.

Effect of different concentrations of Potassium dihydrogen phosphate

The different concentration of KH2PO4 such as 0.9, 1.1, 1.3 (control), 1.5, and 1.7mM
was added to the basal medium minus KH2PO4 and different parameters of the two algae
were recorded.

Effect of different concentrations of Sodium chloride

Different concentrations of NaCl such as 0.08, 0.26, 0.45 (control), 0.60, and 0.80
mM was added to the basal medium minus NaCl and different parameters of the two algae
were recorded.

66
Comparative study of H. lacustris HPI- 001 grown in Modified 3N-BBM+V medium and
Modified HPI- 001A medium

Composition of Modified HPI- 001A medium

Macro elements

NaNO3 - 500 mg
CaCl2.2H2O - 25 mg
MgSO4.7H2O - 75 mg
K2HPO4.3H2O - 75 mg
KH2PO4 - 150 mg
NaCl - 45 mg
Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2MO4.2H2O - 4.0 mg
Na2EDTA - 0.75g
Glass distilled water - 1000 mL
Vitamin B1 - 1.2 mg
Vitamin B12 - 10 µg
pH - 7.5

In the present attempt the above medium of Modified HPI- 001A was formulated
based on the observation made on growth characteristics of H. lacustris HPI- 001 grown in
different conditions of the basal medium.

The alga, H. lacustris HPI- 001 was grown in the Modified HPI- 001A medium for a
period of 30th days and the different parameters were recorded at an interval of every
5 days and compared with the alga grown in the basal medium

67
Comparative study of H. lacustris SAG- 19a grown in Modified 3N-BBM+V medium
and Modified SAG-A medium

Composition of Modified SAG- A medium

Macro elements

NaNO3 - 1000 mg
CaCl2.2H2O - 25 mg
MgSO4.7H2O - 75 mg
K2HPO4.3H2O - 100 mg
KH2PO4 - 200 mg
NaCl - 45 mg
Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2MO4.2H2O - 4.0 mg
Na2EDTA - 0.75g
Vitamin B1 - 1.2 mg
Vitamin B12 - 10 µg
Glass distilled water - 1000 mL
pH - 7.0

The above medium of Modified SAG- A was formulated based on the observation
made on growth characteristics of H. lacustris SAG- 19a grown in different conditions of
Modified 3N-BBM+V medium.

The alga, H. lacustris SAG- 19a was grown in the Modified SAG- A medium for a
period of 30th days and the different parameters were recorded at an interval of every
5 days and compared with the alga grown in the basal medium.

68
Heterotrophic mode on growth, carotenoids and astaxanthin production of two
strains of Haematococcus lacustris

Effect of different concentration of Sodium acetate

The basal medium was added with different concentrations of CH 3COONa such as
0.25, 0.5, 1.0, 1.5 and 2.0 mM and recorded different parameters of the two test algae.

Effect of different concentration of Glucose

The basal medium was added with different concentrations of C6H12O6 such as 0.25,
0.5, 1.0, 1.5 and 2.0 mM and recorded different parameters of the two test algae.

Effect of different concentration of Sucrose

The basal medium was added with different concentrations of C12H22O11 such as
0.025, 0.05, 0.100, 0.250, 0.500 and 1.0 mM and recorded different parameters of the two test
algae.

Mixotrophic mode on growth, carotenoids and astaxanthin production of two

strains of Haematococcus lacustris

Effect of different concentration of Sodium acetate

The strains were also allowed to grow under mixotrophic condition in the basal
medium was added with different concentrations of CH3COONa such as 0.25, 0.5, 1.0, 1.5
and 2.0 mM under 30 µEm-2 s-1 at 12/12 light/dark cycle and recorded different parameters of
the two test algae

Effect of different concentration of Glucose

The basal medium was added with different concentrations of C6H12O6 such as 0.25,
0.5, 1.0, 1.5 and 2.0 mM and recorded different parameters of the two test algae.

69
Effect of different concentration of Sucrose

The basal medium was added with different concentrations of C12H22O11 such as
0.025, 0.05, 0.100, 0.250, 0.500 and 1.0 mM and recorded different parameters of the two test
algae.

Effect of different commercial fertilizers on H. lacustris

Effect of different concentrations of N: P: K (17: 17: 17)

The basal medium minus NaNO3, K2HPO4.3H2O, and KH2PO4 was amended with
different concentration of commercial fertilizer N: P: K such as 0.25, 0.50, 0.75, 1.0, 1.25 and
1.50 g/L and recorded different parameters of the test two algae.

Effect of different concentration of Urea

The basal medium minus of NaNO3 was added with different concentrations of
commercial fertilizers urea (CH4N2O) such as 0.10, 0.20, 0.26, 0.30, 0.40 and 0.50 g/L and
recorded different parameters of the two test algae.

Effect of different concentrations of DAP and Potash

The two strains grown in the basal medium minus NaNO3, K2HPO4.3H2O
and KH2PO4 amended with different concentrations of commercial fertilizer DAP
(Diammonium phosphate) + Potash : 0.10 + 0.05, 0.15 + 0.10, 0.20 +0.15, 0.25 + 0.20 and
0.30 + 0.25 g/L and recorded different parameter.

Effect of different concentrations of food grade Sodium bicarbonate

The basal medium was added with different concentrations of sodium bicarbonate
such as 0.3, 0.6, 0.9, 1.2, 1.5, and 1.8 mM and recorded different parameters of the algae.
The organism was grown in the basal medium treated as control.

70
Effect of different concentrations of food grade Magnesium sulphate

The basal medium devoid of MgSO4 was supplemented with different concentrations
of commercial MgSO4 such as 0.25, 0.50, 0.75, 1.0, 1.25 and 1.50 g/L and recorded different
parameters of the test organisms. The alga grew in the bold basal medium considered as
control.

Comparative study of H. lacustris HPI- 001 grown in Modified 3N-BBM+V medium and
Modified commercial HPI- 001B medium

Composition of Modified commercial HPI- 001B medium

Macro elements

N:P:K (17:17:17) - 0.50 g

CH4N2O - 0.30 g
CaCl2.2H2O - 2.5 g
MgSO4.7H2O - 0.75 mg
(NH4)2HPO4+KCI - 0.15+0.10 g
NaCl - 45 mg
Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2MO4.2H2O - 4.0 mg
Na2EDTA - 0.75 g
Glass distilled water - 1000 mL
pH - 7.5

In the present attempt the above medium of Modified commercial HPI- 001B was
formulated based on the observation made on growth characteristics of H. lacustris
HPI- 001 grown in different conditions of the basal medium.

71
 The alga, H. lacustris HPI- 001 was grown in the Modified commercial HPI- 001B
medium for a period of 30th days and the different parameters were recorded at an interval of
every 5 days and compared with the alga grown in the basal medium (3N-BBM+V)

Comparative study of H. lacustris SAG- 19a grown in Modified 3N-BBM+V medium

and Modified commercial SAG- B medium

Composition of Modified commercial SAG- B medium

Macro elements

N:P:K (17:17:17) - 0.50 g

CH4N2O - 0.40 g
CaCl2.2H2O - 2.5 g
MgSO4.7H2O - 0.75 mg
(NH4)2HPO4+KCI - 0.15+0.10 g
NaCl - 45 mg
Trace elements
FeCl3.6H2O - 97.0 mg
MnCl2.4H2O - 41.0 mg
ZnCl2.6H2O - 5.0 mg
CoCl2.6H2O - 2.0 mg
Na2MO4.2H2O - 4.0 mg
Na2EDTA - 0.75 g
Glass distilled water - 1000 mL
pH - 7.5

The above medium of Modified commercial SAG- B was formulated based on the
observation made on growth characteristics of H. lacustris SAG- 19a grown in different
conditions of Modified 3N-BBM+V medium.

The alga, H. lacustris SAG- 19a was grown in the Modified commercial SAG- B
medium for a period of 30th days and the different parameters were recorded at an interval of
every 5 days and compared with the alga grown in the basal medium

72
Astaxanthin estimation

The amount of astaxanthin was estimated from the acetone extract of the algal sample
at 480 nm using an absorption coefficient, A% of 2500 by following the method as described
by Davies (1976)

A480 × Volume of sample × Dilution factor × 10

Astaxanthin (mg/L) =
2500

Extraction of total carotenoids from Haematococcus lacustris HPI- 001

Ten litre of 30th day old red culture of Haematococcus lacustris HPI- 001 grown in
the Formulated Enriched Modified 3N-BBM+V medium under culture condition was taken
and centrifuged at 5000 rpm for 15 min and the supernatant was decanted.
The pellet was homogenized in a mortar and pestle using 100% acetone in the ratio of 1:10
w/v and then centrifuged at 5000 rpm for 10 min. The supernatant containing the pigments
was retained and the pellet was again homogenized in fresh solvent and subsequently
centrifuged this process until the pellets colourless. The combined crude extracts were
concentrated in a rotary evaporator at 60°C. The sample was a reddish brown colour and the
yield of total carotenoids was subjected for both TLC and Column chromatography.

Spectral analysis of astaxanthin

Thin Layer Chromatography (TLC)

The total carotenoids extracted from the strain of Haematococcus lacustris

HPI- 001 was loaded on a precoated TLC plates and kept in the TLC jar had different
proportions of acetone and hexane in the ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1
in the order to select a suitable solvent system for good separation of pigments on TLC.

73
Column chromatography

Column chromatography was performed in 60 cm × 2 cm glass column Silica gel

(MERK 60- 120 meshes or SRL 60- 120 meshes). One hundred mg of total carotenoids of
H. lacustris HPI- 001 was loaded on the top of column and eluted at a rate of 2 mL/min using
the 3:7 ratios of acetone and hexane as a solvent system. 20 mL of each fraction was
collected in the test tube. Each test tube fraction was compared in an authentic astaxanthin
procured from the Sigma, USA, by TLC technique. The astaxanthin fractions of Rf values at
0.37, 0.58 and 0.73 were pooled and further purified using neutral alumina
100 cm × 1 cm glass column using in the 3:7 ratio of acetone and hexane. The purified elute
was concentrated and dried at room temperature. The resultant amorphous reddish dry
powder was used for the following studies.

Ultra Violet (UV) Visible spectroscopic analysis

After chromatography, the areas of TLC plates contained astaxanthin esters were
carefully removed by scraping off the silica at the appropriate Rf values such as 0.57, 0.58
and 0.73 and mixed with acetone and analyzed under UV absorption, in a using a
double-beam Hitachi U2900 UV-Vis spectrophotometer (Hitachi, Japan) and compared with
standard astaxanthin (Sigma Grade) at 480 nm.

Infra-Red spectroscopic analysis

The purified astaxanthin sample was ground with IR grade Potassium bromide (KBr)
(1:10) pressed into discs under vacuum using spectra lab Pelletiser and compared with
authentic astaxanthin. The IR was recorded Shimadzu FTIR 8000 series in the region
between 4000 and 500 cm-1.

1
H Nuclear Magnetic Resonance (NMR) analysis

1
H NMR spectra of the purified astaxanthin sample were recorded at 23ºC in CDCl3
using a BURKER 300 MHz spectrometer and they were assigned by comparison of chemical
shifts and coupling with those of related compounds. Chemical shifts were reported as values

74
δ-relative to tetramethylsilane (TMS) as internal reference and coupling were reported in
Hertz.

13
C Nuclear Magnetic Resonance (NMR) analysis

13
C NMR spectra of the sample astaxanthin were recorded using a BURKER 300
(75MHz) spectrometers in CDCL3 as a solvent and they were assigned by comparison of
chemical shifts and coupling constants δ-relative to tetramethylsilane (TMS) as an internal
reference and coupling constant were reported in Hertz.

Analysis of astaxanthin through HPLC

Astaxanthin sample of H. lacustris HPI- 001 was subjected for HPLC (JASCO HPLC
1500 Series) analysis using Lichrospher C18 100 RP (25.0cm × 4.6mm × 5µm) column, PU
1580 Pump, MD 1515 PDA detector. The following solvent was used at a flow rate of 1.25
ml min-1: (A) acetone and (B) methanol: H2O (9:1 v/v). The separation of astaxanthin was
achieved by the gradient between solvent A and B for 40 min as follows: B was run at 80 to
20% for 25 min, 20% for 10 min, and 20 to 80% for 5 min.
The separated astaxanthin esters were identified using a photodiode array detector
(Brinda et al., 2004). The peaks were integrated at 480 nm quantify free astaxanthin and
astaxanthin esters.

Mass Spectroscopic analysis (MS)

The astaxanthin sample obtained from the Haematococcus lacustris HPI- 001 was
subjected to FAB mass spectra using a JEOL SX 102/DA-6000 Mass Spectrometer/Data
system using Argon/Xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was
10 kV and the spectra were recorded at room temperature.

75
DPPH radical scavenging assay

Principle

The scavenging reaction between (DPPH) and an antioxidant (H-A) can be written as:

(DPPH) + (H-A) DPPH-H + (A)

(Purple) (Yellow)

Antioxidants react with DPPH, which is a stable free radical and is reduced to the
DPPH-H and as a consequence the absorbance decreased from the DPPH radical to the
DPPH-H form. The degree of discoloration indicates the scavenging potential of the
antioxidant compounds or extracts in terms of hydrogen donating ability
(Blois et al., 1958)

Preparation of reagents

2, 2-diphenyl-1-picrylhydrazyl (0.1 mM)

2, 2-diphenyl-1-picrylhydrazyl (DPPH) of 3.9 mg was dissolved in 100 ml methanol.

Procedure

Assessment of scavenging efficacy of purified astaxanthin form on 2, 2-diphenyl-1-

picrylhydrazyl (DPPH) was performed according to the method of Blois (1958) with slight
modification. 1.0 ml of different concentrations of purified astaxanthin between 20 and 120
µg was added to 1.0 ml of methanolic solution of DPPH (0.1 mM). The mixture was
vigorously shaken and incubated in a dark at 37oC for 30 min. The absorbance was read at
517 nm against reagent (1.0 ml of DPPH and 1.0 ml of methanol instead of the sample).
Ascorbic acid was used as a positive control. The percentage inhibition of DPPH radical
scavenging activity was calculated by the following formula.

Percentage DPPH scavenging effect = [(A0 - A1)/A0] × 100

76
 Where A0 - absorbance of the control and A1 - absorbance of the sample

Superoxide radical scavenging assay (Nishikimi et al., 1972)

Preparation of reagents

Phosphate buffer (0.1 mM) pH 7.4

Nitroblue tetrazolium salt (NBT) (150 µM)

NBT of 12.2 mg was dissolved in 100 ml of phosphate buffer of 100 mM at pH 7.4

Nicotinamide adenine dinucleotide (NADH) (468 µM)

NADH of 31 mg was dissolved in 100 ml of phosphate buffer (100 mM) at pH 7.4

Phenelzine methosulphate (PMS) (60 µM)

PMS of 18.3 mg was dissolved in 100 ml of phosphate buffer (100 mM) at pH 7.4.
The superoxide radical scavenging ability of purified astaxanthin was assessed by the method
of Nishikimi et al., (1972). In this study superoxide anion radical was generated in 3.0 ml
reaction mixture containing 1.0 ml of NBT solution (150 µM), 1.0 ml of NADH
(468 µM) and 1.0 ml of different concentrations of purified astaxanthin (20- 120 µg). The
reaction mixture was initiated by adding 1.0 ml of PMS solution (60 µM) to the mixture and
incubated at 37oC for 5 min. The absorbance of the reaction mixture was read at 560 nm
against a blank (treated in the same manner instead of sample 1.0 ml methanol was added).
Ascorbic acid was used as a positive control. The capability of the scavenging of superoxide
anion radical was calculated by the following formula.

Percentage (%) inhibition of SOD scavenging effect = [(A0-A1)/A0] ×100

Where A0 - absorbance of the control and A1 - absorbance of the sample

77
Reducing power assay (Oyaizu, 1986)

Principle

The substance which has reduction potential, react with Potassium ferricyanide (Fe 3+)
to form potassium ferrocyanide (Fe2+) which then reacts with Ferric chloride to form a
ferrous complex that has an absorption maximum at 700 nm.

Antioxidant
Potassium ferricyanide + Ferric chloride Potassium ferrocyanide + Ferrous chloride
Preparation of reagents

Phosphate buffer (0.2 M) pH 6.6

Potassium ferricyanide (1% w/v)

Potassium ferricyanide of 1 g was dissolved in 100 ml double distilled water

Trichloroacetic acid (TCA) (10% w/v)

TCA of 10 g was dissolved in 100 ml of double distilled water.

Ferric chloride (0.1% w/v)

Ferric chloride 0.1 g was dissolved in 100 ml of double distilled water.

The reducing power assay was determined by the following method of Oyaizu,
(1986). Different concentrations of the purified astaxanthin (20 to 120 µg) in 1.0 ml of the
solvent mixture was added with Phosphate buffer (2.5 ml) and Potassium ferricyanide
(2.5 ml) and incubated at 50oC for 20 min. Trichloroacetic acid (2.5 ml) was added to the
mixture which was then centrifuged at 3000 rpm for 10 min whenever necessary.
The upper layer of solution (2.5 ml) was mixed with double distilled water (2.5 ml) and
freshly prepared Ferric chloride solution (0.5 ml). The absorbance was read at 700 nm using

78
distilled water as a blank. Ascorbic acid was used as a positive control. Increase in
absorbance of the reaction mixture indicates an increase in reducing power.

Phophomolybdenum reduction assay (Prieto et al., 1999)

Principle

This assay is based on the reduction of Mo (VI) to Mo (V) by the extract and the
subsequent formation of a green phosphate/Mo (V) complex at acidic pH.

Preparation of reagents

Sulphuric acid (0.6 M)

Sulphuric acid of 3.26 ml was diluted and made up to 100 ml with double distilled
water.

Procedure

The total antioxidant activity of purified astaxanthin was determined by the standard
method (Prieto et al., 1999). The total antioxidant capacity of the extracts at different
concentrations (20-120 µg) was evaluated by the phosphomolybdenum assay based on the
reduction of Mo (VI) to Mo (V) by the extracts and subsequent formation of a green
phosphate-Mo (V) complex in acidic condition. The purified astaxanthin at different
concentrations (1.0 ml each) was added to 3 mL of reaction mixture containing 0.6 M sulfuric
acid, 28 mM Sodium phosphate and 4 mM ammonium molybdate. The content was then
incubated for 90 min at 95°C. After the samples had cooled to room temperature,
the absorbance of the mixture was read at 695 nm against a blank. For reference, the
appropriate solutions of ascorbic acid were used .

79
ABTS+ free radical scavenging activity (Re et al., 1999)

Principle

The principle behind the technique involves the reaction between ABTS and
Potassium persulphate to produce the ABTS+ free radical cation a blue-green chromogen. The
presence of antioxidant-reductant, the colored radical is converted back to colorless ABTS +
and the absorbance of which is read at 734 nm.

Preparation of reagents

ABTS+ Solution
ABTS+ of 360 mg was dissolved in 100 ml of double distilled water

Potassium persulphate (2.45 mM)

Potassium persulphate of 66 mg was dissolved in 100 ml of double distilled water.

Procedure

The ABTS+ assay the stock solution including 7 mM ABTS+ solution and
2.45 mM Ammonium persulphate solution were prepared. The working solution was then
prepared by mixing the two stock solutions in equal quantities and allowing them to react for
12h at room temperature in the dark. The solution was then diluted by mixing 1 ml of ABTS +
solution with 60 ml of methanol to obtain an absorbance of 0.70 units at 734 nm using the
spectrophotometer. A fresh ABTS+ solution was prepared for each assay. Different
concentrations of purified astaxanthin (5-30 µg/mL) was prepared and allowed to react with 1
ml of ABTS+ solution and absorbance was recorded at 734 nm after 10 min using the
spectrophotometer. Ascorbic acid was served positive control.

Percentage (%) inhibition of ABTS activity = [(A0 - A1)/A0] × 100

Where A0 - absorbance of the control and A1 - absorbance of the sample

80
Antiproliferative activity of Haematococcus lacustris HPI- 001 astaxanthin on human
lung adenocarcinoma-A549 cell lines and human metastatic breast cancer
MDA-MB-231 cell lines (in-vitro)

Cell culture reagents

Dulbecco’s Modified Eagle Medium (DMEM)

Commercially available DMEM contained 7.5% Sodium bicarbonate solution was

taken. To 500 mL of DMEM, 5 mL of Penicillin/Streptomycin solution and 0.5 mL of
Amphotericin B solution were added. Then the medium was sterile filtered (0.22 µM) inside
the hood. The medium was then dispensed into a sterile container and stored at 4 oC.

DMEM Medium

Ten g of DMEM was dissolved in 800 mL of sterilized double distilled water. To this
solution 32.5 mL of 7.5% Sodium bicarbonate was added followed by the addition of 10 mL
Penicillin/Streptomycin solution and 1 mL of Amphotericin B solution. The pH was adjusted
to 7.4 using 1 N HCl. The final volume was made up to 1 litre with sterilized double distilled
water. Then the medium was filtered (0.22 µM) inside the hood. The medium was then
dispensed into a sterile container and stored at 4oC.

Growth Medium (DMEM with 10% FBS)

Ten mL of FBS was made up to 100 mL using sterile DMEM. It was stored in a
sterile container in cool and aspectic conditions.

Phosphate Buffered Saline (PBS) (pH 7.4)

Sodium phosphate monobasic (NaH2PO4) of 0.63 g, 0.17 g of Sodium phosphate

dibasic (Na2HPO4) and 4.5 g of Sodium chloride (NaCl) were taken and dissolved in 500 mL
of double autoclaved milliQ water. The pH was then adjusted to 7.4 using 1 N HCl and 1 N
NaOH, sterile filtered (0.22 µM) and then stored in a sterile container.

Trypsin-EDTA versus Glucose Solution

Trypsin was purchased as 1 x with EDTA (0.5% trypsin, 5.3 mM EDTA sodium salt).
(Note: Freeze/thaw process does not affect the enzyme activity). Thawing was done at room
temperature.

81
Physiological Saline (0.89%)

Sodium chloride of 890 mg was dissolved in 100 ml of double autoclaved milliQ

water.

Cell line

Human lung adenocarcinoma-A549 and MDA-MB-231 cell lines were procured from
the National Centre for Cell Science (NCCS, Pune), India. The cells were grown in T75
culture flasks containing DMEM medium supplemented with 10% FBS.
Upon reaching confluence, the cells were detached using Trypsin-EDTA solution.

Passaging of cells

Upon reaching confluence human lung adenocarcinoma cell line A549 and
MDA-MB-231 were passaged as follows.

The medium from the culture flask was aspirated. The flask was rinsed with 2 mL of
PBS and aspirated again quickly. 1.5 mL of the trypsin-EDTA solution was added and
incubated at 37ºC for about 2 min (until cells started detaching). As soon as the cells were
detached, transfer the trypsinized medium containing cells using a transfer pipette into a
15 ml falcon tube and it was centrifuged at 1000 rpm for 5 min. The medium was carefully
aspirated off and care was taken not to put the pipette tip in the bottom of the tube, where the
cells were pelleted. The cells were gently resuspended in the DMEM medium with 10% FBS
by pipetting up and down 5-8 times gently. From the cell suspension, a drop was placed on
the edge of the coverslip of Neubauer hemocytometer. The drop was led to run under the
coverslip by capillary action. Care was taken not to force the liquid and the entry of the air
bubble was avoided. Then the cells from the E1, E2, E3, E4 and E5 squares were counted
under the inverted microscope. The cells were then gently resuspended and transferred to
sterile T 75 culture flasks and the volume of medium was made up to 10 ml with growth
medium per flask.

Testing the viability of cells

The viability of A549 and MDA-MB-231 cells was assessed by trypan blue exclusion
tests the following methods (Perry et al., 1997).

82
Reagents

Trypan blue solution: (0.5% trypan blue (w/v) in physiological saline).

Procedure

Trypan blue solution of 100 l was mixed with 100 l of cells contained in the
medium and incubated for 5 min at 37ºC. The cells were then washed thrice with saline and
10 l of this suspension was placed in a hemocytometer and viewed under a microscope. The
unstained cells represented the viable cells whereas the damaged cells were stained. The
number of stained and unstained cells was counted and the percentage of viable cells was
calculated using the formula:

No. of unstained cells

% of viability = × 100
Total no. of cells

The viability of the cells was found to be between 90-95%.

Cell proliferation assay

The proliferation of A549 and MDA-MB-231 cells was assessed by MTT

(3-4, 5-dimethyl thiazolyl-2-2, 5-diphenyl tetrazolium bromide) method (Pineiro et al., 2007).

Principle

The assay is based on the reduction of soluble yellow tetrazolium salt to insoluble
purple formazan crystals by metabolically active cells. Only live cells are able to take up the
tetrazolium salt. The enzyme (mitochondrial dehydrogenase) present in the mitochondria of
the live cells is able to convert internalized tetrazolium salt to formazan crystals, which are
purple in colour. Then the cells are lysed using 20% SDS solution, which releases the
formazan crystal. These crystals are solubilized by DMF present in the solubilizer. The
colour developed is then determined in an ELISA reader at 620 nm.

83
Reagents

1. MTT (0.5 mg/ml)

MTT of 5.0 mg of was dissolved in 10 ml of serum-free DMEM medium.

2. Solubilizing solution (20% Sodium Lauryl Sulfate (SDS) in 50%

Dimethylformamide DMF)
Five mL DMF was made up to 10mL with double distilled water to which 2.0 g of
SDS was added and mixed well.

3. Phosphate Buffered Saline (PBS; pH 7.4).

Procedure

Carcinoma cell lines of A549 and MDA-MB-231 cells were plated in 24 well plates at
a concentration of 5 × 104 cells/well 24 after plating, cells were washed twice with 500 µl of
serum-free medium and starved by incubating the cells in serum-free medium for an hour at
37ºC. After starvation, cells were treated with 10, 25, 50, 75, and 100 μg/mL of astaxanthin
obtained from H. lacustris HPI- 001 at different concentrations for 24 hours. At the end of
treatment, the medium from control and purified astaxanthin treated cells were discarded and
500 µl of MTT containing DMEM (0.5 mg/ml) was added to each well. The cells were then
incubated for 4 h at 37ºC in the CO2 incubator.

The MTT containing medium was then discarded and the cells were washed with 1x
PBS (1 mL). The crystals were then dissolved by adding 500 µL of solubilization solution
and this was mixed properly by pipetting up and down. Spectrophotometrical absorbance of
the purple-blue formazan dye was recorded in a microplate reader at
620 nm. The OD of each sample was then compared with the control OD and the graph was
plotted. The growth inhibition rate was calculated as follows.

A620/nm of treated cells

% Growth inhibition = × 100
A620/nm of control cells

Control cultures were treated with DMSO. The maximum concentration of DMSO
added to the medium (0.01%). 24 well plates were divided into five sections, with one section

84
being treated by control culture media, the other treated with one of the following 500 μL
each: culture media containing 10, 25, 50, 75, and 100 μg/mL of astaxanthin obtained from
H. lacustris HPI- 001. The cultures were again incubated as above. After 24 h 500 μL of 0.5
mg/mL MTT solution was added to each well and the cultures were further incubated for 4h
and then 500 μL of 37% SDS in 50% dimethylformamide (DMF) was added and the formed
crystals were dissolved gently be pipetting 2 to 3 times.

Ethidium bromide/acridine orange staining (EB/AO staining or dual staining)

EB/AO staining was carried out by the following method (Gohel et al., 1999)

Reagents

1. Ethidium Bromide stock - 1mg of EB in 1 mL of PBS.

2. Acridine Orange - 1mg of AO in 1 mL of PBS.

3. Staining dye mixture - 200 L EB stock + 200 L AO stock + 600 L PBS.

Procedure

A549 and MDA-MB-231 cells were plated at a density of 5 x 104 in 6 well plates
containing sterile coverslips. They were allowed to grow at 37ºC in a humidified CO 2
incubator until they are 70 - 80% confluent. Then cells were treated with purified astaxanthin
(10, 25, 50, 75, and 100 µg/mL) for 24 h. The culture medium was aspirated from each well
and the cells were gently rinsed twice with PBS at room temperature. Then the coverslips
were taken and kept on glass slides and stained with 100 µl of dye mixture (1:1 of EB and
AO), immediately viewed under a fluorescence microscope.

Viable cells had showen green fluorescent nuclei with an organized structure. The
early apoptotic cells had yellow chromatin in nuclei and exhibited highly condensed or
fragmented. Apoptotic cells also showed membrane blebbing. The late apoptotic cells had
orange chromatin with nuclei that were highly condensed and fragmented. The necrotic cells
had bright orange chromatin in round nuclei. Only cells with yellow, condensed, or
fragmented nuclei were counted as apoptotic cells in a blinded, nonbiased manner. For each
sample, at least 500 cells/well and 4 wells/condition were counted, and the percentage of
apoptotic cells was determined

85
 [% of Apoptotic cells = (total number of Apoptotic cells/total number of cells
counted) × 100].

Flow cytometric analysis of cell cycle

Flow cytometric analysis was carried out as described by Giridharan et al. (2002).
Briefly, 1×106 cells were plated in 100-mm Petri dishes with DMEM containing
10% FBS. Cells were incubated for 24 h in 5% CO2 and 95% air at 37°C. Control cells
received 0.1% DMSO containing DMEM, and purified astaxanthin treated cells received 10,
25, 50, 75, and 100 µg/mL of astaxanthin. After 24 h, the cells were trypsinized and
combined with floating cells in the medium they were used for flow cytometry assay. The
treatment protocol is as follows: 1×106 cells were taken from control and from astaxanthin
treated plates and were centrifuged at 1000g for 5 min. The supernatant was removed and
cells were washed twice with PBS. The pellet was resuspended in 500 μl of ice-cold PBS and
cells were mixed by aspiration 20 times using a pipette. Cells were fixed by adding 5 ml of
cold ethanol drop by drop and were kept at –20°C overnight. After overnight fixation, ethanol
was removed by centrifuging at 1000 g for 10 min. The pellet was washed twice with PBS +
1% BSA (ethanol-fixed cells were difficult to pellet; adding BSA or serum to the wash
medium overcame this). The pellet was resuspended in 800 μl of PBS containing 1% BSA.
One hundred microlitres of 10x Propidium iodide solution was added (500μg/ml Propidium
iodide in PBS, pH 7.4) and one hundred microlitres of RNase A was added (10 mg/ml
prepared in 10 mM Tris-HCl, pH 7.5) and incubated at 37°C for 30 min. Cell-cycle analysis
was performed using a Beckman vantage flow cytometer and quantification of cell cycle
distribution was performed using MultiCycle software (Phoenix Flow System, San Diego,
CA, USA). Percentage of cells in the different cell-cycle phases was assessed.

Western blot analysis

Western blot analysis for protein expression in A549 and MDA-MB-231 cells were
assessed by the following method (Ramakrishnan et al., 2009)

Principle
Proteins were extracted from the cells and separated on 12% Polyacrylamide gel on
the basis of size/charge. They are then transferred on to Nitrocellulose membranes. Following

86
the transfer, the protein of interest can be detected by incubation of the membrane in an
antibody solution followed by detection with an enzymatically labeled secondary antibody.
The protein expression can be visualized by the chemiluminescent method.

Reagents

1. Radio immuno precipitation buffer (RIPA) (150 mM NaCl, 50 mM Tris, 1 mM

EDTA, 1% NP 40, 0.5% Sodium deoxy cholate, 0.1% SDS, pH 7.4).

2. 2 x sample buffer with reducing agent (Sample solubilising buffer, SSB)

(125mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% -mercaptoethanol and
0.004% bromophenol blue).

3. 1 x Tris buffered saline (TBS, pH 7.4): 0.2 M Tris in 0.89% Sodium chloride).

4. Wash buffer (TTBS): 0.1% Tween 20 in 1 x TBS.

5. Blocking buffer: 5% non-fat dry milk powder and 0.1% Tween 20 in 1 x TBS.

6. Antibody dilution buffer: 5% BSA and 0.1% Tween 20 in 1x TBS.

7. Nitrocellulose membrane.

8. DAB/developing solution: Freshly prepared in a brown bottle

(5 mg Diaminobenzidine hydrochloride in 10 ml 1x TBS + 5 µl of 30% H2O2).

Procedure

Fifty g protein of the total cell lysate was mixed with an equal volume of 2x sample
buffer, boiled for 5 min at 95ºC, cooled, loaded on each lane of 8-15% polyacrylamide gel,
and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at
room temperature. The resolved proteins were electrophoretically transferred to
Nitrocellulose membranes. The membranes were then blocked in 5% nonfat milk in Tris-
buffered saline with 0.1% Tween-20 for 1 h at room temperature, and probed with the
following primary antibodies: Bcl-2 (rabbit polyclonal antibody at a dilution of 1:500); Bax
(rabbit polyclonal antibody at a dilution of 1:500, active Caspase-3 (goat polyclonal antibody
at a dilution of 1:250); -actin
(mouse monoclonal antibody at a dilution of 1:2000) overnight at 4ºC. The blots were then

87
extensively washed with Tris-buffered saline with 0.1% Tween-20 and then incubated with
respective (anti goat, anti-rabbit and anti mouse) HRP labeled secondary antibody (Genei,
Bangalore, India) at dilution of 1:2000 for 1 h at room temperature.
After extensive washes in TBS-T, the bands were visualized by treating the membranes with
3,3’-diaminobenzidine tetrahydrochloride (SRL, Mumbai, India). The membranes were
photographed and quantitated with image analysis software, NIH, USA. Densitometry data
presented in bar graphs are “fold change” as compared with control in each case.

Statistical analysis

The experiments were carried out in three duplicates. All data obtained were analyzed
by students-test using MS-Excel, represented as mean ± SD. The results were computed
statistically (OriginPro 8 software). The Least Significant Difference (LSD) analysis was
performed to group the treatment values.

88