Shockley1_Protocol
Project
protocol
—
Contents
Workflow
and
sampling
Equipment
Reagents,
supplies,
and
solutions
Procedure:
Blood
chemistry
and
electrolyte
measurement
using
Beckman
CX5
Synchron
Delta
Chemistry
Autoanalyzer
Data
References
Acclimation
to
test
conditions
In
general
all
mice
are
housed
singly
before
they
are
tested.
Workflow
and
sampling
Steps |
|
|
|
|
1 |
Mice
are
fasted:
food
and
water
removed
for
5
hrs,
6-11
am |
- |
- |
- |
2 |
Sample
tubes
prepared
for
blood
collection |
0.5 |
80
samples/batch |
- |
3 |
Mice
weighed
and
blood
collected
via
retro-orbital
vein |
2.5 |
80
samples/batch |
bw |
4 |
Post-blood
collection
clean-up
|
0.5 |
80
samples/batch |
- |
5 |
Blood
samples
centrifuged
|
1.0 |
80
samples/batch |
- |
6 |
Serum
extracted
(frozen
when
stored)
|
1.0 |
80
samples/batch |
- |
7 |
Beckman
CX5
system
calibrated
|
0.25 |
- |
- |
8 |
Reagents
changed
or
replenished
|
0.25 |
- |
- |
9 |
|
0.25 |
- |
- |
10 |
|
2.5 |
15
sectors
(105
samples)/run |
- |
11 |
Serum
HDL
measured
automatically
|
1.0 |
15
sectors
(105
samples)/run |
- |
12 |
Serum
CHOL,
GLU
and
TG
prepared
|
1.0 |
15
sectors
(105
samples)/run |
- |
13 |
Serum
lipids
and
calcium
measured
automatically
|
3.0 |
15
sectors
(105
samples)/run |
Ca,
CHOL,
GLU,
TG,
HDL,
NEFA,
T4,
GLDH,
BUN |
14 |
|
0.5 |
- |
- |
15 |
Computer
printed
data
are
labeled,
data
files
collected
and
stored
on
disks
|
0.5 |
- |
- |
Equipment
and
supplies
- Refrigerated
tabletop
micro-centrifuge:
Eppendorf
Centrifuge,
Model
#
5415C
- Repeat
pipettes
and
regular
Pipetman:
250
µL
and
100
µL,
respectively
- Automated
blood
chemistry
analyzer:
Synchron
CX5
Delta
(Beckman
Coulter,
Inc.,
Fullerton,
CA)
- A
dedicated
DOS-based
desktop
computer
controls
the
programming
of
this
analyzer
- A
dedicated
printer
prints
the
results
as
they
are
measured,
and
an
electronic
file
is
simultaneously
transferred
to
a
second,
Windows-based
computer,
which
stores
the
data
files
- Perfusion
kit:
venous
catheters
or
large
needles
and
5
mL
syringes
Panels
A-E
illustrate
the
Beckman
Synchron
CX5
Delta.
Panel
B
shows
a
closer
look
of
area
1
in
Figure
A.
Figure
C
presents
a
closer
look
of
area
2
in
Panel
A.
Panel
D
depicts
a
closer
view
of
area
3,
where
ancillary
reagents
are
refilled
in
Panel
A.
Panel
E
reveals
the
content
of
area
4,
where
samples
are
set
up
in
trays
for
an
automated
run
in
Panel
B.
For
Synchron
CX5
parts
and
all
consumables
Reagents
and
solutions
- Micro-hematocrit
tubes
(75
µL
capacity)
coated
with
Heparin
and
equipped
with
a
rubber
bulb
expunger
- Heparin
anticoagulant
(Sigma
(Sodium
Salt)
50,000
U
Cat.
#H-3393)
->Heparin
1000
U/mL:
50
mL
of
distilled
autoclaved/sterile
water
+
50,000
U
Heparin
Stock
Solution
- Sterile
physiological
(0.9%)
saline
solution
Expendables:
(1)
1.5
µL
Eppendorf
tube,
(2)
0.5
mL
Beckman
Coulter
Microtube
Tubecup
("sector
cup")
(3)
100
µL
and
250
µL
pipette
tips
(4)
1
cc
syringes
with
needles
Reagents:
The
Chemistry
Analyzer
(or
"CX5")
uses
Beckman
Coulter
three-compartment
reagent
cartridges
for
HDL,
CHOL,
TG,
and
GLU.
Each
cartridge
contains
enough
reagents
for
300
tests
(approximately
104
mL).
In
addition,
in
order
to
run
the
HDL
Cholesterol
test,
HDL
Cholesterol
Separation
Reagent
(15
µL
per
sample)
is
needed.
The
bottle
from
Beckman
Coulter
contains
a
volume
of
34
mL.
If
the
dilution
of
serum
samples
becomes
necessary
due
to
low
serum
volume,
use
0.9%
saline
solution
for
the
dilution.
Reagent: Reorder
No.
Cholesterol
(CHOL)
Reagent
467825
Glucose
(GLU)
Reagent
442640
Triglycerides
(TG)
Reagent
445850
Calibration
Reagents:
The
two
calibration
reagents
are
"Synchron
Systems
HDL
Cholesterol
Calibrator"
(for
HDL
only),
and
"Synchron
Systems
Multi
Calibrator"
(for
CHOL,
GLU,
and
TG).
Controls:
The
controls
for
HDLc
are
Beckman
Coulter
Vigil
Lipid
Control
1
and
Beckman
Coulter
Vigil
Lipid
Control
2.
The
controls
for
CHOL,
GLU
and
TG
are
Synchron
Control
Comprehensive
Chemistry
Control
Serum
Level
1,
Level
2
and
Level
3.
Procedure:
Blood
serum
chemistry
and
electrolyte
measurement
using
Beckman
CX5
Synchron
Delta
Chemistry
Autoanalyzer
I.
Pre-test
feeding
regimen
a.
All
mice
received
standard
diet
of
6%
fat
until
6-wks
of
age.
b.
At
6-wks
of
age,
the
mice
are
divided
into
two
dietary
groups
with
5
males
and
5
females
per
strain
per
group.
c.
The
1st
group
is
given
the
standard
low-fat
diet,
and
the
second
group
is
given
an
atherogenic
high-fat
diet
(30%
Kcal
from
dairy
fat)
containing
by
weight
1%
cholesterol
and
0.5%
cholic
acid
to
increase
cholesterol
uptake.
d.
The
2
dietary
groups
are
then
kept
on
the
feeding
regimen
for
4
consecutive
weeks.
e.
At
10–13
wk
old
and/or
after
4
wks
on
the
dietary
regimen,
the
mice
are
single-housed
for
3
days
before
blood
sampling
(see
below).
f.
On
the
day
of
the
blood
collection,
the
mice
are
placed
in
clean
box
cages
without
food
from
6
a.m.
to
11
a.m.
g.
Following
blood
collection
the
mice
are
subsequently
euthanized
by
cervical
dislocation
between
11
a.m.
to
noon
(to
minimize
circadian
variation
in
mRNA
expression),
and
then
perfused
with
saline
before
the
liver
is
dissected
and
the
median
lobe
is
harvested
and
cut
into
1
cm2
sections
to
be
placed
in
RNAlater®
at
room
temperature
for
24
hrs,
then
stored
at
-80°C
until
RNA
extraction
for
microarray
is
done.
Investigator's
notes:
"Due
to
difficulties
associated
with
obtaining
blood
samples
from
CAST/EiJ
and
PERA/Ei
mice,
ages
at
bleeding
and
death
ranged
from
20
to
55
wks
of
age.
We
removed
these
strains
from
our
primary
analysis
to
eliminate
unwanted
variability
from
this
age
difference.
However,
the
data
are
available
via
the
CGD
website..."
II.
Blood
serum
collection
a.
At
10-13
wks
of
age
and
after
a
5-hr
fast
from
6:00-11:00
a.m.,
each
mouse
is
weighed
and
blood
sample
obtained
between
11:00
a.m.
to
noon
to
minimize
sampling
variability.
b.
Approximately
200
µL
of
blood
is
obtained
from
each
mouse
via
retro-orbital
bleed
using
heparin-coated
Hematocrit
tubes.
Remaining
blood
in
the
Hematocrit
tube
is
flushed
out
using
a
rubber
squeeze
bulb
assembly.
c.
Blood
samples
are
collected
into
pre-labeled
1.5
mL
Eppendorf
tubes
and
momentarily
stored
in
ice
until
all
the
samples
are
ready
to
be
centrifuged.
d.
Blood
samples
are
then
centrifuged
for
5
min
at
14,000
RPM
using
a
refrigerated
table-top
micro-centrifuge
to
separate
the
serum.
e.
The
top
serum
layer
is
pipetted
into
pre-labeled
0.5
mL
Eppendorf
tubes
and
frozen
until
ready
to
be
assayed;
remaining
packed
blood
cell
layer
is
discarded.
f.
Previously
frozen
serum
samples
are
defrosted
at
room
temperature
for
about
30
min
before
measurements
are
done.
NOTES:
Air
bubbles
are
avoided
and
eliminated
during
sample
loading
in
sector
cups
as
they
interfere
with
the
colorimetric
assay.
III.
Using
Beckman
CX5
Synchron
system
to
measure
serum
glucose
and
lipids
chemistries
Panel
F
shows
2
empty
sector
cups.
Panels
G
shows
sector
cups
with/out
hemolyzed
sample,
and
Panel
H
shows
sector
cups
without
and
with
(red
arrow)
air
bubbles.
Panels
I
and
J
show
consecutive
sector
trays
identified
with
bar
codes
and
seven
sector
cups
contained
within
each
tray.
a.
In
preparation
for
the
auto-analyzer,
bar-coded
sectors
with
cups
in
place
are
loaded
with
85
µL
of
completely
thawed
serum
(enough
for
the
direct
measurement
of
CHOL,
GLU,
and
TG).
b.
Up
to
five
sectors
are
manually
placed
on
the
carousel
to
be
run.
Since
each
sector
is
bar-coded,
the
Beckman
automatically
detects
a
sector
that
has
been
run;
regardless,
finished
sectors
are
removed
immediately
as
soon
as
they
come
up.
c.
The
Beckman
CX5
is
operated
according
to
manufacturer's
instructions
in
the
measurement
of
serum
HDL,
CHOL,
GLU,
and
TG,
which
are
run
together.
d.
-Function
key
F1
is
used
to
deploy
"Sample
Program",
Sample
type
"1"
is
for
serum.
-Function
key
F2
is
used
to
deploy
"Program
Batch/Sector(s)"
and
"sector(s)
to
program:"
(i.e.
sectors
1-5
is
programmed).
When
"Batch"
mode
activated,
7
cups
possible.
"Number
of
cups
in
batch:"
message
is
displayed,
the
total
cups
for
this
batch
is
then
(7
x
5)
=
35
cups
(the
maximum
number
of
cups
that
can
be
run
in
a
batch
is
98).
To
program
remaining
sectors,
F2
Program
Batch
is
deployed
again.
-Panel
"12"
is
preprogrammed
for
CHOL/GLU/TG
and
"HDLD"
is
manually
"Selected"
from
the
screen
menu.
Once
the
correct
chemistries
are
selected,
they
are
"ENTERed"
to
bring
about
"SAMPLE
TYPE",
wherein
"1"
is
given
to
denote
serum
(not
plasma).
-Function
key
F8
is
used
to
set
up
the
programmed
batches
and
to
advance
to
the
next
cup/sample
(from
cup
#1
up
to
cup
#7)
in
a
given
sector.
By
selecting
F8
ID
numbers
can
be
recorded
and
reviewed
against
an
Excel
reference
sheet.
Notes:
Since
ID
sample
numbers
or
field
identifiers
are
un-editable
once
Beckman
CX5
is
in
operation,
relevant
information,
including
sample
type
(i.e.
plasma
vs
serum),
dilution
factor,
and
other
information
must
be
precisely
entered.
•
As
soon
as
six
sectors
are
programmed,
the
<prev
screen>
is
activated
first,
and
then
the
Master
Screen,
and
then
last
START
(green)
button.
The
Beckman
automatically
starts
sampling
the
first
five
programmed
sectors.
Additional
sectors
can
be
programmed
while
the
Beckman
is
operating.
•
In
the
event
that
the
Beckman
alarm
is
activated
because
of
reagent
volume
getting
low,
<prev
screen>
is
activated
to
turn
off
the
alarm
and
make
the
necessary
notations
for
the
record.
The
next
available
reagent
cartridge
is
automatically
installed.
•
When
all
the
data
is
safely
recorded,
Function
key
F5
is
used
to
clear
all
the
information
regarding
a
sector
after
ENTERing
the
number
of
the
sector
to
be
deleted.
e.
Once
a
sector
is
finished
running,
the
results
are
automatically
printed,
removed
from
the
printer,
and
then
labeled
accordingly.
Otherwise,
the
printed
paper
is
checked
and
guarded
from
rolling
back
into
the
printer
and
disrupting
the
data
recording.
f.
Used
Eppendorf
tubes,
pipette
tips,
sector
cups,
and
reagent
cartridges
are
discarded
into
biohazard
waste
containers,
and
any
spilled
liquids
are
cleaned.
Definitions
&
formulas
The
average
of
the
diluents
analyzer
value
is
subtracted
from
the
analyzer
value
multiplied
by
two
(dilution
factor)
to
obtain
the
HDL
(indirect)
values.
HDL
(indirect)
values
=
(sample
analyzer
value
x
2)
-
average
of
diluents
analyzer
value
Data
collected
by
investigator
Cohorts:
control
diet
high-fat
diet
difference*
- blood
chemistry
- calcium
(Ca)
- glutamate
dehydrogenase
(GLDH)
- glucose
(GLU)
- blood
urea
nitrogen
(BUN)
- blood
lipids
- HDL
cholesterol
(HDL)
- total
cholesterol
(CHOL)
- nonesterified
fatty
acid
(NEFA)
- triglycerides
(TG)
- body
weight
- endocrine
hormones
*MPD
Note:
"Difference"
vectors
were
computed
by
MPD
from
the
Shockley1
strain
means
(high
fat
diet
over
control),
and
are
available
for
download
in
a
separate
supplementary
file.
|
|
Test age:
10-13wks
