Vulpe1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedures
Definitions
and
calculations
Data
References
Workflow
and
sampling
Workflow
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Mice
are
fed
purified
diet
until
8-wk
of
age |
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Mice
are
euthanized
without
fasting |
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Blood
is
collected
via
cardiac
puncture |
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Serum
is
collected
and
stored
for
later
analysis |
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Liver
is
harvested
and
stored
for
later
analysis |
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Liver
is
processed
for
measuring
mineral
content |
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7 |
Iron,
copper,
and
zinc
levels
in
the
liver
are
measured |
Mass
spectrometer |
iron,
copper,
and
zinc
levels |
8 |
Serum
is
analyzed
for
diferric
transferrin |
Gel
electrophoresis
system |
serum
diferric
transferrin |
9 |
Total
serum
transferrin
levels
are
assessed |
Western
blotting
system |
(total
serum
transferrin) |
Equipment
Reagents,
supplies,
solutions
•
AIN93G
purified
diet
containing
~35
ppm
iron
(Dyets)
•
carbon
dioxide
gas
•
5
mL
syringes
•
centrifuge
tubes
•
microcentrifuge
tubes
•
liquid
nitrogen
•
suprapure
nitric
acid
•
metal-free
water
•
polyclonal
antibody
to
human
transferrin
(1
in
1,000
dilution;
Silenus
Laboratories,
Hawthorn,
Australia)
Procedures
I.
Measurement
of
serum
transferrin
levels
a.
Mice
are
fed
a
purified
diet
containing
~35
ppm
iron
ad
libitum
until
8
wk
of
age
before
testing
begins.
b.
To
facilitate
the
collection
of
blood,
mice
are
euthanized
with
carbon
dioxide
gas
without
fasting.
c.
Blood
is
collected
by
cardiac
puncture
using
19G
needle
and
5
mL
syringe.
d.
Collected
blood
samples
are
then
kept
at
room
temperature
for
at
least
30
min
and
allowed
to
settle.
e.
To
collect
serum
samples,
clotted
blood
samples
are
centrifuged
at
1,000
g
for
15
min.
f.
Serum
samples
are
then
aliquoted
into
microcentrifuge
tubes,
snap-frozen
in
liquid
nitrogen,
and
stored
at
-70°C
for
subsequent
analysis.
g.
To
determine
serum
diferric
transferrin
levels,
urea
polyacrylamide
gel
electrophoresis
system
is
used.
h.
To
evaluate
total
transferrin
levels
in
the
same
serum
samples,
Western
blotting
is
used
with
a
polyclonal
antibody
to
human
transferrin
that
cross-reacts
with
the
mouse
protein.
II.
Measurement
of
liver
mineral
content
a.
Liver
tissues
are
harvested,
snap-frozen
in
liquid
nitrogen,
and
stored
at
-70°C
for
subsequent
analysis.
b.
To
test
for
liver
mineral
content,
liver
tissues
are,
first,
vacuum-dried
overnight
using
a
freeze-drying
system.
c.
Dried
liver
samples
are
weighed,
and
then
digested
in
suprapure
nitric
acid
using
a
microwave
digestor
(CEM
system
5).
d.
Digested
liver
samples
are
then
diluted
with
metal-free
water
to
give
a
final
nitric
acid
concentration
of
~1
M
of
nitric
acid.
e.
In
order
to
measure
levels
of
iron,
copper,
and
zinc
in
the
liver,
an
inductively
coupled
plasma-atomic
emission
spectrometry
is
used.
f.
The
metal
content
of
the
liver
is
expressed
as
micrograms
per
gram
(µg/g)
dry
weight
of
tissue.
All
samples
are
measured
in
triplicates.
Data
collected
by
investigator
•
Liver
copper
(Cu),
iron
(Fe),
and
zinc
(Zn)
contents
(dry
weight)
•
Plasma
diferric
transferrin
as
a
percent
of
total
transferrin
Definitions
transferrin:
serum
glycoprotein
that
binds
to
iron
reversibly
to
control
levels
of
circulating
free
iron.
diferric
transferrin:
form
of
transferrin
that
reversibly
binds
two
iron
ions
(the
majority
of
circulating
iron).
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Test age:
8wks
