Role and mechanism of microglial activation in
iron-induced selective and progressive dopaminergic neurodegeneration
Supplemental Material
Table of Contents
Methods
Primary midbrain neuron-microglia-astroglia cultures
Primary microglial cultures
Immunostainings
O2.- assay
iROS assay
Methods
Primary midbrain neuron-microglia-astroglia cultures
Rat primary midbrain neuron-microglia-astroglia cultures were prepared from the brains of
embryonic day 14 ± 0.5 days of SD rats and 13 ± 0.5 days of mice, following our previously described
protocol (Zhang et al. 2007). The ventral midbrain tissues were removed and dissociated by mild
mechanical trituration. Cells were seeded at 5 × 10 5/well for rats and 1 × 106/well for mice to a 24-well
culture plate precoated with poly-D-lysine ( 20 μg/mL) and maintained at 37°C in a humidified
atmosphere of 5% CO2 and 95% air in 0.5 mL/well maintenance medium. The medium consisted of
minimum essential medium (MEM), containing 10% heat-inactivated fetal bovine serum (FBS) and
10% heat-inactivated horse serum (HS), 1 g/ L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate,
100 μM non-essential amino acids, 50 U/mL penicillin, and 50 μg/mL streptomycin. Three days after
the initial seeding, 0.5 mL of fresh maintenance medium was added to each well. Seven-day cultures
were used for the treatment. The composition at the time of treatment was about 48% astroglia, 11%
microglia, and 40% neurons, of which about 2-3 % was TH-immunoreactive (TH-ir) dopaminergic
neurons. Seven days after the treatment, immunostainings with anti-TH, Neu N and OX-42 were
performed.
Primary microglia cultures
Primary mixed glia cultures with a purity of >98% were prepared from the whole brains of pups
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from 1-day-old SD rats, NOX2+/+ and NOX2-/- mice, following our previously described protocol
(Zhang et al. 2007). Brain tissues were triturated after removing meninges and blood vessels. Cells
were seeded at 5×107 for rats and 1×108 for mice in a 150 cm2 culture flask. After a confluent
monolayer of glia had been obtained, microglia were shaken off for 1 h at 180 rpm. Microglia were
seeded at 1 × 105/well in a 96-well culture plate for measuring the levels of extracellular superoxide
(O2.-) and intracelluar reactive oxygen species (iROS), and at 2 x 106/well in a 6-well culture plate for
detecting gene and protein expressions of NOX2 subunits P47 and gp91, PKC-σ, P38, ERK1/2, JNK
and NF-КBP65 24 h after seeding.
Immunostainings
TH-ir dopaminergic neurons and total neurons were stained with anti-TH (1:20,000) and anti-Neu
N (1:1,000) antibodies, respectively, whereas microglia were stained with anti-OX-42 antibody (1:500).
Formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogen peroxide (15 min) followed by
sequential incubation with blocking solution (20 min), primary antibody (4°C, overnight), biotinylated
secondary antibody (2 h), and ABC reagents (2 h). Color was developed with 3, 3’-diaminobenzidine.
For the morphological analysis, the images were recorded with an inverted microscope connected to a
charge-coupled device camera operated with MetaMorph software. For visual enumeration of the
immunostained dopaminergic neurons in primary midbrain neuron-microglia-astroglia cultures, total
TH-ir dopaminergic neurons in a 24-well plate were counted under the microscope at ×100
magnification. For determining the numbers of Neu N-ir total neurons and OX-42-ir microglia in
primary midbrain neuron-microglia-astroglia cultures, 9 representative areas per well of the 24-well
plate were counted by two individuals in a blind fashion under the microscope at ×100 magnification.
Three wells were in each group and results were from 3 independent experiments in triplicate and
expressed as the percentage of the vehicle-treated control group and were the mean ± SE (Zhang et al.
2007).
O2.- assay
Microglial production of extracellular O2.- was determined by measuring the SOD-inhibitable
reduction of tetrazolium salt WST-1. Primary microglia cultures were seeded at 1 × 10 5 /well and
grown for 24 h in a 96-well culture plate in MEM containing 10% FBS. For the O 2.- assay, the cultures
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were washed two times with Hank’s balanced salt solution (HBSS) without phenol red. Cultures
received 100 L/well of Fe2+ in HBSS followed by 100 L /well of 4 mM WST-1 in HBSS with or
without SOD (250 U/well), in a final concentration of 5, 25 and 100 M. The absorbance at 450 nm
was read for a period of 40 min at 37°C. The amount of O 2.- production was measured by the increased
absorbance in 30 min and expressed as the percentage of control group (Zhang et al. 2007).
iROS assay
iROS was determined by using a DCFH-DA assay as described previously with modification
(Zhang et al. 2007). DCFH-DA enters cells passively and is deacetylated by esterases to
non-fluorescent DCFH. DCFH reacts with ROS to form DCF, the fluorescent product. DCFH-DA was
dissolved in methanol at 10 mM and was diluted 500-fold in phenol red-free HBSS, containing 2%
FBS and 2% HS, to give a final concentration of 20 M. Primary microglia cultures were seeded at 1 ×
105 /well and grown for 24 h in a 96-well culture plate in MEM containing 10% FBS. For the iROS
assay, the cultures were washed two times with HBSS without phenol red. 100 L/well DCFH-DA was
added to the cultures and incubated for 2 h at 37°C. Then 100 L /well of Fe2+ with indicated
concentrations of 5, 25 and 100 M were added to the cultures. Fluorescence intensity was measured at
485 nm for excitation and 530 nm for emission. iROS production was measured by the increased
absorbance in 2 and expressed as the percentage of control group.
REFERENCE
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