Materials and Methods

"Everyone has a will to win but very few have the will
to prepare for win"
-Vinci Lombardi
Materials and Methods
3.1) Chemicals
Acrylamide, 7-aminoactinomycin D (7-AAD-lOJ..LM), 4-aminobenzoic acid
hydrazide (ABAH-lOOJ..LM), ammonium persulfate (APS), anti rabbit lgG HRP,
apocynin (5mM), aproteinin (lJlg/ml), ascorbic acid (lmM), bisacrylamide, p
mercaptoethanol CPME), bovine serum albumin (BSA), bromophenol blue (BPB),
calmodulin (CaM), carboxy PTIO (1 OOJlM), comassie brilliant blue R-250 (CBB),
deoxyribonucleic acid (DNA), 3,3-Bis( aminoethyl)-1-hydroxy-2-oxo-1-triazene
(DETA-NO), 5,6-diaminofluorescein diacetate (DAF-DA:10J..LM), 2',7'-
dichlorofluorescein diacetate (DCFDA-1 OJ..LM), dihydroehidium (DHE-1 Of.!M),
dihydrorhodamine 123 (DHR123-5f.1M), diphenyleneiodonium chloride (DPI,lOf.lM),
dithiothreitol (DTT-1mM), ethylenediamine tetra acetic acid (EDTA-lmM), ethylene
glycol-bis(P-amino ethyl ether), N,N,N',N'-tetraacetic acid (EGTA-1mM),
histopaque -1119, -1083 and 1077, hoechst 33342, horse radish peroxidase (HRP-
lOunits/ml), leupeptin (1JlM), LR white, magnesium chloride (MgCh), mercuric
chloride (HgCh-lOOJlM), N-acetyl-L-cysteine (NAC, 5mM), N-[2 hydroxyethyl]
piperazine N' [2-ethanesulfonicacid] (HEPES), nicotinamide adenine dinucleotide
reduced (NADH), nicotinamide adenine dinucleotide phosphate reduced (NADPH),
7-nitroindazole (7-NI-1mM), N"'-nitro-L-arginine methyl ester (L-NAME, 1JlM),
nitrosocysteine (S-NO) antibody, nitrotyrosine (Y-N0
2
) antibody, nonidet (NP-40),
osmium tetroxide (Os0
4
), paraformaldehyde (PFA-4%), pepstatin A (20Jlg/ml),
percoll, phenyl methyl sulfonyl fluoride (PMSF-100Jlg/ml), phorbol12-myristate 13-
acetate (PMA-50nM), pluronic F127, poly L-lysine, ponceau S, potassium hydrogen
phosphate monobasic (KH
2
P0
4
), potassium hydrogen phosphate dibasic (KzHP04),
propidium iodide (PI-5Jlg/ml), S-nitroso N-acetyl penicilamine (SNAP), sodium azide
(NaN
3
), sodium chloride (NaCl), sodium fluoride (NaF-lmM), sodium hydrogen
phosphate monobasic (NaH
2
P0
4
), sodium hydrogen phosphate dibasic (NazHP04),
sodium nitrite (NaN0
2
), sodium nitrate (NaN0
3
), sodium nitropruside (SNP), sodium
orthovenadate (Na
3
V0
4
-2mM), sodium pyruvate (lOOmM), soyabean trypsin
inhibitor, teleost fish gelatin, TEMED, 5,5',6,6'-tetrachloro-1,1',3,3'-
tetraethylbenzimidazolcarbocyanine iodide (JC-1 ), 5,6, 7,8-tetrahydrobiopterin (BH
4
-
1mM), trisodium citrate, triton X 100, trypan blue (2mg/ml), tween 20 were obtained
Role ofnitric oxide in neutrophil maturation and function 52
Materials and Methods
from Sigma Aldrich Co. (MO, USA). Uranyl acetate was purchased from Polaron
Equipment Ltd. (Watford, England).
Table 6: Composition of various reagent/buffers
Reagent/Buffer Composition (mM)
PBS NaCI 138 mM; KCI 2.7 mM; Na: HP0
4
4.3 mM;
KH
2
P0
4
1.5 mM (pH 7.4)
HBSS NaCI 138 mM; KCI 2.7 mM; Na
2
HP0
4
8. 1 mM;
KH
2
P0
4
1.5 mM; Glucose 10 mM. (pH7.4)
TBST Tri s 25mM, NaCI 150mM, tween 20 (0.1 %)
TE 1 OmM Tris (pH 8.0) and lmM EDT A (pH 8.0) mixed,
autoclaved and stored at 4C.
Blocking buffer
Griess reagent
4% PFA
Lysis Buffer
RIPA Buffer
Sodium Citrate
Hypotonic Propidium
iodide solution
Staining Buffer
Incubation buffer
Stop buffer
. coli (FITC)
Culture medium
Binding buffer
activity
PBS containing 1% BSA, 3% gelatin and 0.05%
Tween-20
0.1% aquous N-(1-naphthyl)-ethylenediamine
dihydrochloride 1% sulfanilic acid in 2.5 %
phosphoric acid
4% Paraformaldehyde in PBS (pH 7.4)
NaCI lOOmM, Tris HCI lOmM pH 7.4, EDTA 1mM
pH 7 .4, aprotinin lf.Lg/ml, phenylmethylsu1fonyl
fluoride (PMSF) 1 OOf.Lg/ml, Pepstatin 20f.Lg/ml,
Sodium orthovenadate (Na
3
V0
4
) 2rnM
PBS contammg 0.1% SDS, 0.5% sodium
deoxycholate, 1% Triton X-1 00, and protease
inhibitors
3 f1 f (Pfl; 6 5")
50f.Lg/ml with 0.03% NP-40 in 0. 1% sodium citrate
PBS containing 1% BSA
Hepes 25 mM; NaCI 140 mM; KCI 5.4 mM; MgCI
2
I
mM; pH 7.4
NaCI 118mM, KCI 4.7mM, KH
2
P0
4
1.18mM,
NaHC0
3
1 mM, EDTA4 mM, N"'-Nitro-L-arginine
methyl ester (L-NAME) 2 mM; pH 5.5.
Heat inactivated at 60C for 30 minutes, labeled with
FITC (50 =Yml) in the dark at room temperature for
I hr with continuous shaking, washed twice in HBSS.
RPMI-1640 containing NaHC0
3
. 2mM glutamine,
10% (v/v) FBS, 100 units/ml penicillin and IOOf.Lg/ml
streptomycin
Hepes-NaOH 10mM (pH 7.5), NaCI 140mM, 2.5mM
CaCI2
HEPES 150 mM ; pH 7.4, NaCl450 mM, KCI 150
Functions
Multiple
Neutrophils
Western blotting
RNA .
lmmunolabelling
Nitrite
estimation
Fixati on
Western Blotting
lmmuno
prec ipitati on
Blood isolation
Cell cycle
CD marker
labeling
NOS acti vity
NOS activity
Phagocytosis
Cell culture
v Annex in
labeling
Caspase activity Caspase
buffer
TEM Fixative
mM, EGTA 1.2 mM, 1.5%, Nonidet P40 0.3%
CHAPS, 30% sucrose, 30 mM DTT, 3 mM PMSF
Paraformaldehyde 2% and glutaraldehyde 2.5% in LR white
0. 1M cacodylate buffer (pH 7.4). fixative
Blocking solution PBS containing 0.1% BSA (w/v), 0.1% (v/v) Teleost Imrnuno-gold
_____ and 0.05% v/v Tween-20 labelling
Monoclonal anti- CDll b- Pacific blue (Pac Blue)/ phycoerythrin (PE), anti-
CD15- Allophycocin (APC), anti- CD16-FITC, anti - CD34-PE and anti- CD45-PE
conjugated antibodies were purchased from Becton Dickinson (CA, USA). Polyclonal
Role of nitric oxide in neutrophil maturation and.fimction
53
Materials and Methods
nNOS, iNOS, MPO, Lactoferrin were procured from Santa Cruz (CA, USA).
Elastases, actin, anti-goat/mouse IgG HRP were purchased from Sigma (MO, USA).
Anti Rabbit/mouse/goat IgG alexa flour 488/594 antibodies were purchased from
Molecular probe .Cyclin B, D, E and CDK2, 4, 6 antibodies were purchased from Cell
Signaling Technology. Hybond P, ECL film, Ecews was purchased from
Amersham biosciences (Upsala, sweden). BCA protein assay kit was purchased
from Pierce (Rockford, IL, USA). All other chemicals used in the present study were
of analytical grade and were procured from SRL, Bombay (India).
3.2) Animals and human blood donors
Male Sprague dawley rats (200-250 g) were obtained from the National Laboratory
Animal House of the Institute. They were housed in polypropylene cages and
provided with chow pellets and water ad libitum. All the animal experiments were
conducted according to the ethical guidelines of the IAEC. Blood was collected in
citrate-phosphate-dextrose (CPD) as anti coagulant in the ratio of 1:7 from healthy
human volunteers after a written consent from the donors at the blood donation centre
ofSanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow. Bone marrow
cells were obtained by aspiration from the posterior superior iliac crest of healthy
volunteers and CML patients after informed consent had been given at Sanjay Gandhi
Postgraduate Institute of Medical Sciences, Lucknow. A detailed medical history and
physical examination was carried out before phlebotomy.
3.3) Isolation of human and rat PMN s
Rat blood (Sethi et al., 1999) was collected under ether anesthesia by cardiac puncture
in sodium citrate (0.129 M, pH 6.5, 9:1 v/v) and human blood (Brinkmann et al.,
2004) was collected from the anticubital vain. Platelet rich plasma was removed by
centrifugation at 1000 rpm for 20 min at 20C (Beckman TJ6, USA) and remaining
blood was centrifuged at 2000 rpm for 20 min at 20C, platelet poor plasma was
discarded and the huffy coat was subjected to dextran sedimentation (6% w/v) for 1 h
at 3 7C (Boyum, 1968). Upper leukocyte rich layer was transferred to a fresh tube and
centrifuged at 2000 g for 20 min at 20C (3K30 Sigma Centrifuge, Germany). Pellet
was suspended in 2 ml Hank's balanced salt solution (HBSS) [Composition (mM):
NaCl 138; KCI 2.7; NazHP04 8.1; KHzP0
4
1.5; Glucose 10; pH 7.2-7.4] and loaded
Role ofnitric oxide in neutrophil maturation andjimction 54
Materials and Methods
on the top of the 2 ml Histopaque density gradient (1119 and 1083) and centrifuged at
700xg for 30 min at 20C. PMNs rich layer was recovered from the interface of
Histopaque 1119 and 1 083 and contaminating RBCs were removed by hypotonic
lysis. The cells were washed and suspended in HBSS. PMNs were counted in
Neuber's chamber and the viability of the cells was determined by Trypan blue dye
exclusion.
Purity of isolated PMNs was checked by immunolabeling using anti-rat CD
11 b-FITC, anti-rat CD 45-PE monoclonal antibodies for 30 min at 4C in dark. After
incubation cells were washed twice with HBSS and suspended in 0.5 ml of 1%
paraformaldehyde containing 0.1% sodium azide and kept at 4 oc till analysis by flow
cytometer (F ACS Calibur, Becton Dickinson, USA) using appropriate compensation.
3.4) Isolation of neutrophil precursor cells from rat bone marrow
Neutrophil precursors from bone marrow were isolated using Percoll gradient (1.065,
1.080 and 1.086) according to method described by Cowland and Borregaard (1999).
After centrifugation bone marrow cells were separated into three bands having
myeloblasts (MBs)/Promyelocytes (PMs), Myelocytes (MCs)/metamyelocytes (MMs)
and band cells (BCs)/segmented neutrophils (SCs) in band 3, 2 and band 1
respectively.
Percoll gradient Percoll stock (%) lOx PBS(%) TDW(%)
Density (Density= 1.13) (Density = 1.056)
1.065 45.7 10 44.3
1.080 57.2 10 36.8
1.086 61.8 10 28.2
3.4.1) Cell viability
Rat or human cells (1x10
6
cells/ml) were pre-incubated for 5 minutes with propidium
iodide (PI, 5J.tg/ml) followed by treatment with DETA-NO (1 f.lM to 1 mM) or SNAP
(1 JJM to 1mM) or with various inhibitors used in the study as mentioned in legends
for 30 min at 37C to assess their effect on the cell viability. HL-60 (1x10
6
cells/ml)
control cells or treated with NO-donors in culture medium were taken out and
directly incubated with propidium iodide (PI, 5)Jg/ml) to avoid any further viability
loss during centrifugation, washing. Samples were examined by acquiring 10-20,000
cells after 10 min of staining which were subsequently analyzed by Cell Quest
program (F ACS Calibur, Becton Dickinson, USA) (Saini et al., 2006).
Role ofnitric oxide in neutrophil maturation andjimction 55
Materials and Methods
3.4.2) Immunolabeling of surface markers
Lineage specific CD markers on cell surface offer a wide range of choices to identify
the cell of interest from a mixed population of cells. Mature neutrophils express
CDllb and CD-45 as surface markers. 2x10
6
neutrophils were suspended in lOO!Jl
HBSS and incubated with S!Jl of anti mouse CDllb-PE and anti mouse S!Jl ofCD45-
FITC monoclonal antibodies for 30 minutes at 4C. Matched isotype controls (anti-
mouse IgG-PE and anti mouse IgG-FITC) were also put up simultaneously. Following
incubation the cells were washed twice with 0.5ml of 1% paraformaldehyde
containing 0.1% sodium azide and kept at 4C till analysis (Sharma et al., 2003;
Sharma et al., 2004). Before analysis cell suspension was shaken properly and
acquired by flow cytometer (F ACS Calibur,Becton Dickinson, USA).
3.4.3) Cell sorting by using FACS-Aria
Different neutrophils precursors were sorted according to CD maker 11 b, CD 15 and
CD16 by using FACS-Aria. Band 3 (Myeloblast and promyelocyte) cells were
CD15+ and CDllb- CD marker and sorted at high speed 70!JM nozzle. CD15+,
CDllb+ and CD16- CD marker cells were used as Band 2 (Myelocytes and
metamyelocytes). While mature cells (Bandt, Band cells and Segmented Neutrophils)
were CD 11 b + and CD 16- and used for sorting criteria (Theilgaard-Monch et al.,
2005).
3.5) NO level/ NOS activity assessment in Neutrophils/ Precursors
3.5.1) Nitrite estimation
Nitrite content in the rat neutrophils and precursors was measured by the Griess
reagent. Cells were sonicated on ice and centrifuged at 2000g for 20 min at 4C, the
supernatant thus obtained was treated with cadmium chloride to reduce nitrate to
nitrite to calculate total nitrite. Samples were treated with Griess reagent and
incubated for 30 min at 37C. Concentration of nitrite was estimated by measuring the
absorbance at 548nm using sodium nitrite as standard (Seth et al., 1994).
3.5.2) Nitric oxide synthase activity
NO production was assessed by the formation of L-eH] citrulline from L-eH]
Arginine. Rat neutrophils and precursor cells were sonicated in (lx10
7
cells) in
incubation buffer (Hepes 25 mM; NaC1140 mM; KC15.4 mM; MgCh 1 mM; pH 7.4)
and then incubated in presence of co-factors BH4 (10!JM), NADPH (lmM), FAD
Role of nitric oxide in neutrophil maturation andjimction 56
Materials and Methods
(5!lM), FMN (25!-lM), and calmodulin (lO!lglml) in presence or absence of CaCb
(2mM) and EGTA (5mM) as indicated. Reaction was initiated by the addition of L-
eH] arginine and was continued for 30 minutes at 37C. Reaction was stopped by
addition of ice-cold stop buffer (NaC1,118mM; KC1,4.7mM; KH
2
P04,1.18mM;
NaHC0
3
,1 mM; EDTA,4 mM; Nw-Nitro-L-arginine methyl ester (L-NAME),2 mM;
pH 5.5). The mixture was passed through Dowex 50WX (T-400) columns.
Radioactivity in the eluent was measured using beta scintillation counter (LKB
Wallace 1409, Liquid Scintillation counter, USA) as described by Saini et al.,(2006).
NO synthesis in the neutrophil lysate has been reported as pmol of L-eH] citrulline
formedl30 min/10
7
cells.
3.5.3) NO generation by flow cytometry
Flow cytometry offers an effective tool in studying real time generation of NO in live
cells. Rat and human neutrophils and precursors (2x10
6
cells/ml) were incubated with
4,5-diamino fluorescein diacetate (DAF-2DA,lO!lM) for 5 min and then treated with
BH4 (lmM) with ascorbate (lmM) as indicated for 30 min at 37C. DAF-2DA reacts
rapidly and irreversibly with NO to produce a highly reactive fluorescent product
triazolo fluorescein (DAF-2T). Fluorescence of 10,000 cells was acquired by gating
the neutrophil population and was analyzed by Cell Quest program to determine the
mean fluorescence using F ACS Cali bur, Becton Dickinson, USA (Sharma et al.,
2002)
3.6) Functional studies/activities
3.6.1) Free radical Generation
Rat or human PMNs (2x10
6
cellslml) were incubated with vehicle or various
inhibitors for 5 min at 37C and subsequently loaded with 2' ,7' -dichlorodihydro-
fluorescein diacetate (DCF-DA, 10!lM), dihydrorhodamine-123 (DHR-123, 5!lM) or
dihydroethidine (DHE, 1 OJ..LM) for 5 min, and finally sodium nitroprusside (SNP, 100
11M) I S-nitroso-N-acetyl-penicillamine (SNAP, 10011M) I phorbol 12-myristate 13-
acetate (PMA, 50nM) was added to the suspension. Each sample was monitored for
free radical generation by acquiring 10,000 cells which were subsequently analyzed
by Cell Quest program (F ACS Calibur, Becton Dickinson, USA) (Sharma et al.,
2002).
Role of nitric oxide in neutrophil maturation and jimction 57
Materials and Methods
3. 6.2) Phagocytic activity
Phagocytosis was measured m neutrophils usmg flow cytometry as previously
described by Bazzoni et al.,(1991). E.coli were heat inactivated at 60C for 30
minutes, labeled with FITC (50!-lg/ml) in the dark at room temperature for 1h with
continuous shaking. Labeled bacteria were washed twice in PBS. Neutrophils were
incubated in presence of bacteria at 1:50 as mentioned for 30 minutes, and analyzed
immediately by flow cytometry. To differentiate between phagocytosed and adherent
bacteria Trypan blue (20!-ll of a stock solution of 2mg/ml for 5minutes) was added to
the suspension to quench the fluorescence of the adherent bacterial population and
reanalyzed on flow cytometer (F ACS Calibur, Becton Dickinson, USA).
3.6.3) Myeloperoxidase activity
MPO activity was evaluated following the method of Graff et al.,(1998). Neutrophils
were freeze-thawed consecutively for three times and then sonicated in three cycles of
10 seconds each at 95 watts (W-385 Heat System, Ultrisonics Inc.). Hexadecyl-
trimethyl ammonium bromide (HTAB) was incubated to the cell lysate at 37C for 30
minutes and centrifuged at 3000xg for 20minutes at 20C. Supernatant was taken for
evaluation of enzymatic activity. Enzyme kinetics was run for 3 minutes at 15 sec
intervals at 463 nm on a double beam spectrophotometer (Systronics, India). The
volume of phosphate buffer (Na
2
HP0
4
-50mM; NaH
2
P0
4
-50mM pH6.0) and cell
supernatant with different amount of enzyme content (50-200!-ll) was made upto
2.4ml. To this 500!-ll of 0-dianisidine (200!-lM) and 100!-ll of H
2
0
2
(150!-lM) was
added at 37C. Optical density was recorded at 463nm and was converted to units of
concentration by using molar extinction co-efficient for oxidized o-dianisidine E
=10062[M X cmr
1
Activity ofMPO has been expressed as !lM/min/10
7
cells.
3.6.4) Elastase activity
The elastase assay using N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline
(Meo-Suc-Ala-Ala-Pro-ValpNA) as a substrate was conducted according to Schorr et
al., (2005). Briefly the release of the enzyme was quantified by the photometric
measurement of p-nitroaniline (pNA), the coloured product of the cleavage of Meo-
Suc-Ala-Ala-Pro-ValpNA by elastase. The absorbance of the supernatant was
measured at 405nm.
Role ofnitric oxide in neutrophil maturation andfimction 58
Materials and Methods
3. 6. 5) Immunofluorescence assessment of NETs
Neutrophils and precursor cells Ox10
6
), plated on glass bottomed cover-slips (pre-
coated with 0.001% poly L-lysine) were treated with NO donor, SNAP (100-500!-LM),
PMA (1 0-50nM) or vehicle for 30-180 min in a C0
2
incubator (RS Biotech, UK) at
37C. Effect of various interventions such as N-acetyl-L-cysteine (NAC, 5mM), 7-
nitroindazole (7-NI, 1mM), diphenyleneiodonium chloride (DPI, 10!-LM) or 4-
aminobenzoic acid hydrazide (ABAH, 1 00!-LM) was also monitored on NETs release
by pre-incubating PMNs for 5 min at 37C and then continuous presence with SNAP
for 3 hr. After fixation with 4% Paraformaldehyde, samples were stained overnight
with 20 !lg/m1 of rabbit po1yclonal elastase (Calbiochem, USA) or 5 1-lg/ml mouse
monoclonal histone (Chemicon, UK) antibody and were visualized after treatment
with the secondary antibody (1 :200, chicken anti-rabbit/mouse AF 488 antibody,
Molecular Probes, USA) by confocal microscope (Brinkmann et al., 2004) and
assessed for the incidence of NETs formation (Buchanan et al., 2006). DNA was
stained with propidium iodide (PI, 20!lg/ml), Hoechst 33342 (3!lg/ml) or Sytox green
(5!lM).
High magnification and low magnification images were captured by Carl Zeiss
(Germany) or Olympus (Japan) confocal microscopes by using appropriate lenses and
filters. The mean fluorescence intensities were quantified using LSM Software
Version 4.2 (Carl Zeiss, Germany).
3. 7) Cell growth/proliferation studies
3. 7.1) Cell culture
HL-60 cells/ PMNs were grown in RPMI-1640 containing NaHC0
3
, 2mM glutamine,
10% (v/v) FBS, 100 units/ml penicillin and 100!-lg/ml streptomycin at 37C in a 5%
C02 incubator. HL-60 cells used for experiments were always in log phase. Cells
were culture for various time intervals with NO donors Deta-NO O!lM-1mM), SNAP
0!-LM-1mM), SNP 0!-LM-1mM), NaN02 (1!-LM-mM) or NaN03 (lmM) in C02
incubator.
3. 7.2) Cell synchronization
Serum starvation was used to arrest cells in Go phase of cell cycle by culturing cells 2
days in RPMI-1640 supplemented with 0.5% FCS. Mimosine was also used to cell
Role o.fnitric oxide in neutrophil maturation andfimction 59
Materials and Methods
synchronization at Go phase as described earlier (Krude, 1999). Mimosine stock
solution was prepared at 10 mM in RPMI and filtered through 20-mm filters.
Synchronization ofHL-60 cells by mimosine was achieved by adding mimosine from
the stock solution directly to the cells at the concentrations specified. Cell
synchronization was determined by flow cytometry of isolated nuclei stained with
propidium iodide (5 mg/ml in PBS containing 0.4% Triton X-100) and analyzed by
F ACS calibur (Becton Dickinson).
3. 7.3) Cell growth assessment
The in vitro growth effect ofDETA-NO on the HL-60 cells was determined by trypan
blue dye exclusion. HL-60 cells were grown at 37C in RPMI medium supplemented
with 10% fetal bovine serum, penicillin (1 00 U/ml) and streptomycin sulphate
(1 00!-lg/ml) in a humidified atmosphere of 5% C02. Cells were seeded at a
concentration of 2x10
5
cells/ml and were maintained for logarithmic growth by
passaging them every 2-3 days and incubated for 1-2 days with DETA-NO at various
concentrations. Cells were loaded on a hemocytometer, absorbance at 630 nm, and
viable cell number was determined based on exclusion of trypan blue dye (Lee et al.,
2007).
3. 7. 4) Cell cycle analysis
Cell cycle staining was done according to Krishan(1975) in control and NO donors
treated cells . HL-60 cells (1 x 1 0
5
) were centrifuged at 150g for 5 min and the cell
pellets resuspended in hypotonic Propidium iodide solution (50!-lg/ml with 0.03% NP-
40 in 0.1% sodium citrate). Samples were examined after 5-l 0 min of staining at 4C.
the DNA content of these cells was measured with a flow cytometer (F ACS Calibur,
Becton Dickinson) by using the Cell Quest program, and then the cell-phase
distribution was analyzed by Modfit software (Verity Software, Topsham). This was
method of choice because of its less time required, less sample loss during
centrifugation, high quality CV and less aggregates formation.
3. 7.5) BrdU incorporation
DNA synthesis was assessed by measunng the incorporation of 5-bromo-2_-
deoxyuridine (BrdU) into DNA using a cell proliferation kit (BrdU FITC Kit; BD
Biosciences) according to the manufacturer's instructions. Briefly cells were pulsed
with 1 01-1M BrdU for 30 min in culture followed by fixation, permeabilization, DNase
Role ofnitric oxide in neutrophil maturation andfimction 60
Materials and Methods
treatment and finally detection with anti BrdU-FITC antibody. DNA was
simultaneously stained with 7-AAD. Cells were analysed with FACS-calibur at FL1
and FL3 channels.
3. 7. 6) 3H-thymidine incorporation
HL-60 cells were cultured in RPMI-1640 medium containing 10% FBS. Next day
HL-60 cells (1x 10
5
cells) in RPMI-1640 with 10% FBS were cultured in 96-well
plates, NO donor DETA-NO was added to each well at different concentration from 1
J.!M- 1 mM. 1 J.!Ci of
3
H thymidine was added and culture in 5% C02 at 3 7 C. After
24, 48 and 72 h later, the wells were harvested with a Skatron semiautomatic cell
harvester, and the radioactivity was quantified with a scintillation counter. All the
experiments were done in triplicates.
3.8) Apoptosis assay
3.8.1) Annexin V-PI double staining by flow cytometry
For apoptosis assay, double staining with FITC-labelled Annexin V for phosphatidyl
serine exposed to the outer leaflet of the plasma membrane and PI for cellular DNA
was examined by Annexin V Fitc kit (BD biosciences) according to manufacture's
instruction. Briefly, cells were resuspended in 1 OOJ.!l of binding buffer (1 OmM Hepes-
NaOH,pH 7.5; 140mM NaCL; 2.5mM CaCb) and incubated in the dark at room
temperature for 15minutes in presence of 5 J.!l of annexin V and PI each. Following
incubation 400J.!l of binding buffer was added to each sample and subsequently
analyzed after 10 min by flow cytometry (F ACS calibur, Beckman-Dickinson)
(Vermes et al., 1995).
3.8.2) Evaluation of mitochondrial membrane potential (A film) using JC-1
1 x 10
5
cells were resuspended in RMPI 1640 supplemented with 10% FBS and
stained with 2.5 Jlg/mL JC-1, as previously described. An incubation of 10 min at
room temperature was followed. Cells were then washed and the pellet was
resuspended in PBS for flow cytometric analysis. The color of JC-1 changes from
green to greenish orange as the mitochondrial membrane becomes more polarized.
Green emission was analyzed in FL1 and greenish orange emission in FL2. In healthy
cells, the dye stains the mitochondria bright red. In apoptotic cells, the mitochondrial
membrane potential collapses, and since JC-1 cannot accumulate within the
Role ofnitric oxide in neutrophil maturation andfimction 61
Materials and Methods
mitochondria it remains in the cytoplasm in a green fluorescent monomeric form.
Apoptotic cells, showing primarily green fluorescence, are easily differentiated from
healthy cells which show red and green fluorescence. A decrease in FL-2 fluorescence
is represented as loss in mitochondrial transmembrane potential as discussed
in the results section (Lugli et al., 2005).
3.8.3) TdT enzyme dependent dUTP nick end labelling (TUNEL)
5x10
5
cells grown in RMPI 1640 supplemented with 10% FBS with or without NO
donors were fixed with 4% PF A for 20 min on ice and washed with PBS twice at
300g for 5 min, then pellets were resuspended in chilled 70% ethanol for overnight.
Cells were labelled with APO-Direct Kit, single step method for labelling DNA
breaks with Fitc-dUTP. Briefly, Experimental and positive cells were washed twice
with PBS at 300g for 5min. cells were incubated in staining buffer containing TdT
enzyme, Fitc dUTP for 60 min at 37C. Finally cells were stained with PIIRNase
solution for 30 min. Samples were acquired at F ACS Calibur (Beckman-Dickinson),
with in 2 h of labelling.
3.8.4) Fluorometric Caspase assay
Caspase-3 activity was measured in HL-60 cells at 24 hrs (Cid et al., 2003) after NO
donor treatment. Treatment was given in 100 ofRPMI for indicated time in 96 well
culture plates and finally 50 of3 x lysis buffer (150 mM HEPES, pH 7.4, 450 mM,
NaCl, 150 mM KCl, 1.2 mM EGTA, 1.5% Nonidet P40, 0.3% CHAPS, 30% sucrose,
30 mM DTT, 3 mM PMSF), which included substrate Acetyl-Asp-Glu-Val-Asp-7-
amino-4 methyl-coumarin (DEVD-AMC, Protein concentrations were
measured by BCA Protein Assay Reagent Kit (Pierce) in another set of experiment.
Fluorescence of cleavage of DEVD-AMC into 7-amino-4 methyl-coumarin (AMC)
(Sigma-Aldrich) was measured at 30C using wavelength 380 nm (excitation) and 460
nm (emission) by fluorimeter (Varian Caryeclipse).
3.9) Determination of intracellular GSH
Cels are stained with monobromobimane mBrB ( for 10 mm at room
temperature and run on the flow cytometry with excitation at 353 to 361 nm and
emission at 450 nm (Hedley and Chow, 1994). An increase in fluorescence is core
related with GSH content. A replicate sample was depleted of GSH by treatment with
Role ofnitric oxide in neutrophil rnaturation andjimction 62
Materials and Methods
100 11M N-ethylmaleimide to give a measure of non-specific binding of the mBrB
probe. One major advantage of this assay is that it is a quick and simple measure of an
important oxidant defence system which can be performed on a very small number of
cells. Samples were acquired at FACS Aria (Beckman-Dickinson) and analyzed by
F ACS Diva software (Beckman-Dickinson).
3.10) Expression studies
3.1 0.1) Immunoprecipitation
The PMNs (5x10
7
/ml) were lysed in radio immuno precipitation (RIP A) assay
buffer [PBS containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-1 00, and
protease inhibitors (as in protein extraction buffer)] for 15 min. The supernatant was
precleared with protein A/G agarose (Amersham biosciences, Upsala, Sweden) and
incubated with 1flg primary antibodies (nNOS, iNOS and eNOS); after 2 h, 20111
protein A/G agarose was added and incubated for 1 h. The beads were washed,
resuspended in gel loading buffer, denatured at 95C for 5 min, and subsequently
analyzed by western blotting.
3.10.2) Western blotting
5x10
6
cells were suspended in protein extraction buffer [O.lM NaCl, 0.01M Tris HCl
pH 7.4, 0.001M EDTA pH 7.4, 1flg/ml aprotinin, 100flg/ml phenylmethylsulfonyl
fluoride (PMSF), Pepstatin 20!-lg/ml, Sodium orthovenadate (Na
3
V0
4
) 2mM, DFP
1mM] for 30 min on ice (Saini et al., 2006). Centrifuged at 13000 rpm, 20 minutes, 4
C. Protein concentrations were measured by BCA Protein Assay Reagent Kit (Pierce).
Samples containing equal amounts of protein in Laemmli buffer were separated by 8-
12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes
(Amersham). Nonspecific binding was blocked overnight with TBST [Tris 25mM,
NaC1150mM, tween 20 (0.1 %)] containing 5% (w/v) BSA at 4 C. Membranes were
incubated with nNOS ( 1:1 000), iN OS (1: 1000), nitro-tyrosine (1: 1 000), nitro so
cysteine (1:1000), cyclin B, D1, cyclin E, CDK2, CDK4, CDK6, p21waf/cipl,
p27kip 1 (1: 1 000) in TBST (0.1% v/v) with 5% BSA for 3 hours at RT with, After 5
washes with PBST the membranes were probed with HRP-labeled secondary antibody
(1: 10000 dilution with TBST with 5% BSA) for 2 hours at RT. Membranes were
Role ofnitric oxide in neutrophil maturation andjimction 63
Materials and Methods
washed 10 times with TBST. Proteins were detected using ECL detection kit and
visualized on Hyperfilm (Amersham Biosciences, UK).
3.10.3) RT-PCR analysis
3.10.3.1) RNA isolation
RNA was isolated from neutrophils and its precursors by Tri reagent using modified
method of Chomczynski and Sacchi, (1987). Briefly, cells (5x10
6
) were lysed in 0.8
ml of tri reagent by repeated pipetting and kept at room temperature for 5 min.
Chloroform (160 J..ll) was added and shaken vigorously for 15 seconds followed by an
incubation of 3 min at room temperature to precipitate the proteins. The samples were
centrifuged at 12000g for 15 minutes at 4C (Biofuge Stratos, Heraeus, Germany).
The aqueous phase containing RNA was aspirated into a different tube and
isopropanol (400J..ll) was added to precipitate the RNA. The samples were incubated
for 10 min at room temperature and subsequently centrifuged at 12000g for 15 min at
4C. RNA pellet was washed with 70% alcohol by centrifuging at 7500g for 5min at
4C. The RNA pellet was finally resuspended in 20-30 J..ll DEPC water.
The quality of RNA was assessed electrophoretically on denaturing formaldehyde
agarose gel (Genei) and spectrophotometrically with GeneQuant Pro. Briefly, 3-4 J..ll
of RNA sample was mixed with 10 J..ll formamide (Sigma), 2 J..ll; 5X formaldehyde gel
running buffer and 3.5 J..tl formaldehyde. It was heated at 65C for 15 min and
immediately placed on ice. After 5 min, 2 J..ll formaldehyde gel loading dye was added
followed by 0.5 J..ll EtBr (10 mg/ml stock). The contents were mixed properly and
loaded on to gel for electrophoretic separation. RNA samples with approximately 2:1
ratio of 28S: 18S rRNA and 260/280 values > 1. 7 were used for eDNA labeling
reaction. The quantity of RNA was determined by measuring absorbance (OD) at 260
nm. Where, 1 OD= 40 J..lglml; for single stranded RNA.
3.10.3.2) eDNA synthesis
eDNA was synthesized using first strand eDNA synthesis kit (Fermentas, Canada) as
per manufacturer's protocol .In brief the reaction mixture for eDNA was prepared as
follows:
Template RNA
Oligo dT primer
DEPC treated water
5 J..lg
2 J..ll
up to 12 J..ll
Role of nitric oxide in neutrophil maturation andjimction 64
Materials and Methods
The reagents were mixed gently and spun down for 3-5 second in a micro
centrifuge. The mixture was incubated at 70 C for 5 min, chilled on ice and
drops were collected by light centrifugation
The tubes was placed on ice and the following component were added in the
indicated manner
1 Ox R T buffer 2 )ll
dNTP mix ( 1 0 mM) 4 )ll
RNAse inhibitor 1 )ll
MMLV -RT 1)11
The reagents were mixed gently and drops were collected by light
centrifugation
The mixture was incubated at 42 C for one hour.
The reaction was stopped by heating at 92 C for 10 min and then chilled on
ice. The eDNA was now ready for PCR.
3.10.3.3) Primers
To explore different isoforms of NOS in neutrophils and precursor cells, gene specific
primers were used. Primers (oligos) used for polymerase chain reaction were procured
from Sigma (USA). Following primers were used for the present study-
All the PCR reactions were performed in 0.2 ml transparent PCR tubes (Axygen)
using thermal cycler (Applied Biosystem, Foster city, CA). The PCR reagents
Role of nitric oxide in neutrophil maturation and jimction 65
Materials and Methods
including PCR buffer, nucleotide mtx (dNTPs) and Taq DNA Polymerase were
procured (Promega and Sigma) and used for the standardization and amplification of
the DNA samples. Gradient PCR reactions were performed for each primer pairs and
annealing temperature was optimized.
Reaction components Stock concentration Final concentration Volume
Taq Polymerase Buffer
lOX lX 5.0!11
with MgC1
2
(25mM)
Forward Primer (FP) 1 OOpmol/ 111 15pmoll!ll 3.0!11
Reverse Primer (RP) 1 OOpmol/ 111 15pmoll!ll 3.0!11
Genomic DNA lOOng/111 lng/111 1.0!11
dNTPsmix 2mM 20011M 1.0!11
Taq DNA Polymerase 3U/Ill 0.030/!ll 0.25!11
Sterile water (MQ) - - Up to 5 0 ~ t l
Thermal cycling parameters consisted of 10 min initial denaturation at 95C followed
by 30-35 cycles of 1min denaturation at 94C, 1 min at annealing temp and 1 min
extension at 72C. Final extension at 72C for 4 min was allowed before holding the
reaction at 4C for 30 min. Total 8)!1 of amplified product was mixed with 1/5th
volume of gel loading buffer (analytical grade water containing 30% glycerol, 0.25%
bromophenol blue, 0.25% xylene cynole) and resolved on 1.2% agarose gel in TAE
buffer at 80 volts for 1 hour. DNA markers (100bp, New England Biolabs) were run
along with amplified product. PCR product were resolved on agarose gel containing
0.5)..lg/ml ethidium bromide using horizontal submarine gel electrophoresis apparatus
(Genei, India) and visualized in a UV Transilluminator (Uvitec, UK). The
electrophoresis PCR products were analyzed on gel document system (Amersham
Biosciences, Sweden).
3.1 0.4) Immuno-cytochemical Detection of nitro-tyrosine, nitro so-cysteine
The cells were fixed in suspension with 1% methanol-free formaldehyde in PBS for
15 min on ice followed by suspension in 70% ethanol according to Tanaka et al.,
(2006). The cells were washed twice in PBS and suspended in 0.2% Triton X-100
(Sigma) in a 1% (w/v) solution of bovine serum albumin (BSA; Sigma) in PBS for 30
min to suppress nonspecific antibody (Ab) binding. The cells were then incubated in
1 00)!1 of 1% BSA containing 1:100 diluted anti nitro-tyrosine rabbit Ab or 1:100
Role of nitric oxide in neutrophil maturation and jimction 66
Materials and Methods
diluted anti nitroso cysteine rabbit Ab (Sigma). The cells were then incubated
overnight at 4C, washed twice with blocking buffer, and resuspended in 100 1-11 of
1: 100 diluted Alexa Fluor-488 conjugated F( ab )2 fragment of goat anti mouse IgG
(Molecular Probes, Eugene) and for 60 min at room temperature in the dark. The cells
were then counterstained with 10!-lg/ml 7-AAD in PBS for 20 min at 37C. Cellular
fluorescence was measured using a Cell Quest program (F ACS Calibur, Becton
Dickinson, USA)
3.11) Cytometric bead array and Elisa
Cells supernatants were used to screen cytokine by BD TM CBA human inflammation
kit according to manufacturer instructions. Briefly; 50!-ll of each sample was mixed
with 50!-ll of capture beads and 50!-ll of PE detection reagents. The samples were
incubated at room temperature for 3 h in dark. The samples were washed once and
resuspended in 300!-ll ofwash buffer before acquisition on a FACS Calibur. Data were
analyzed using F ACS Diva software (BD Pharmingen). The range of detection was
20-5000pg/ml for each cytokine measured by CBA. Further quantitative
measurements of different cytokine including Interleukin-8 (IL-8), IFN-y, T G F ~ and
TNF-a were done by using BD Elisa kits according to manufacturer instructions.
Briefly, cell supernatant after DETA-NO treatment were incubated in Capture
antibody coated Elisa plates and cytokine levels were estimated by detection antibody
and HRP substrate. Quantitation was done according to standard provided with kit.
3.12) Confocal microscopy
To explore the intracellular distribution of NOS isoforms and elastase in Neutrophils
and precursors, cells were fixed in 4% (w/v) paraformaldehyde in PBS (pH 7.4) at
25C for 30 min and washed twice for 5 min each with PBS containing 0.5% (w/v)
glycine. The washed cells were allowed to adhere on 0.01% (w/v) poly-L-lysine
coated coverslips, permeabilized with 0.2 % (v/v) Triton X-100 (5-10 min) and
blocked with 10% (v/v) goat serum in PBS for 30 min. Cells were incubated
overnight at 4C with monoclonal antibodies against iNOS/ nNOS/ eNOS and elastase
at a dilution of 1 :200 and subsequently stained with alexa flour 488 or 594 secondary
antibodies (1 :500), at 4C for 4 h in dark. nuclei were stained with Hoechst 33342 dye
(3J.!g/ml) or propidium iodide (PI, 5!-lg/ml) at 25C for 15 min. Cover slips were
mounted in the mounting medium (Oncogene, CA, USA) and images were acquired
under NIKON Eclipse TE 300 Confocal Microscope using 63 x 1.4 NA Plan
Role ofnitric oxide in neutrophil maturation andjimction 67
Materials and Methods
Apochromate lens. Red, green and blue images were collected separately with
excitations at 647 nm, 488 nm, and 405 nm respectively. Adobe Photoshop 7
software was used for further analysis and presentation of images. Control samples
were processed similarly as mentioned above, omitting the primary antibodies (Saini
et al., 2006).
3.13) Immuno-gold electron microscopy
Neutrophils and precursors were fixed with 2% (w/v) paraformaldehyde and 0.5%
(v/v) glutaraldehyde in PBS (pH 7.4) overnight at 4C. Fixed cells embedded in 4%
agarose were cut into smaller pieces. The excess fixative was quenched with 0.5%
(w/v) glycine in PBS (pH 7.4) for 10 min. and they were subsequently thoroughly
washed with PBS containing 0.5% (w/v) glycine at 4C for 10 min. They were
dehydrated in ascending series of ethanol, impregnated in LR-White resin and then
polymerized at 60C for 48 h (Klinger et al., 1997). Ultrathin sections (70-1 00 nm)
were cut and collected on nickel grids. Sections were first blocked in PBS containing
0.1% BSA (w/v), 0.1% (v/v) Teleost Fish gelatin and 0.05% (v/v) Tween-20 for 30
min, and then incubated with monoclonal antibodies against iNOS/nNOS/eNOS at
1 :200 dilutions in the blocking buffer for 4h. After washing five times with blocking
buffer, the sections were incubated 10 nm gold-coupled anti- rabbit/mouse IgG (1:20)
at 37C for 2 h. For negative controls incubation with the primary antibodies was
omitted. The labeled sections were contrasted with uranyl acetate and examined under
FEI Philips (Tecnai-12) and Hitachi (H7500) Transmission Electron Microscopes at
80kV (Saini et al., 2006). Control samples were separately incubated with normal
rabbit and mouse sera in place of primary antibodies.
3.14) Statistical analysis
Data are represented as Mean SEM, of at least 3-5 independent experiments and
were analyzed by one way ANOV A test followed by Newman-Keuls post analysis.
Student's t-test analysis was also used in comparing the Control Vs treated as
specified in legends. Data were considered significant at p<0.05.
Role o_fnitric oxide in neutrophil maturation andjimction 68