ab118183 –
Fatty Acid Oxidation
Human Flow Cytometry Kit
Instructions for Use
For measuring in very long chain specific
acyl-CoA dehydrogenase, medium-chain specific
acyl-CoA dehydrogenase and long-chain
3-hydroxyl-CoA dehydrogenase
This product is for research use only and is not
intended for diagnostic use.
Version 3 Last Updated 29 February 2016
1
Table of Contents
1.
Introduction
3
2.
Assay Summary
5
3.
Kit Contents
6
4.
Storage and Handling
6
5.
Additional Materials Required
7
6.
Preparation of Reagents
7
7.
Sample Preparation
8
8.
Assay Procedure
9
9.
Data Analysis
11
10. Assay Performance and Specificity
12
11. Troubleshooting
16
2
1. Introduction
Principle: ab118183 is a panel of antibodies against key enzymes of
the mitochondrial fatty acid oxidation pathway for flow cytometry.
The assay combines the power of single cell analysis obtained from
flow cytometry with the specificity of antibody-based immunostaining
to quantify protein levels in cultured cells. Cells are harvested and
fixed/permeabilized in suspension, targets of interest are detected
indirectly
with
highly
specific
well-characterized
monoclonal
antibodies that are then labeled with fluorescent antibodies
Background: The Fatty acid B-oxidation (FAO) pathway is a key
metabolic pathway that plays an important role in energy
homeostasis particularly in organs such as the liver, heart and
skeletal muscle.
Oxidation of fatty acids occurs inside the
mitochondria where acyl-CoA esters (activated fatty acids) of various
lengths are shortened into units of acetyl-CoA each time a cycle is
fully completed. Each unit of acetyl-CoA is then oxidized by the
mitochondria into CO2 and H2O via the citric acid cycle and the
mitochondria respiratory chain. The FAO pathway consists of a four
step process which involves: (1) dehydrogenation, (2) hydration, (3)
second dehydrogenation and (4) thiolytic cleavage.
This kit
measures the levels of three of the most studied enzymes in this
pathway: ACADVL, ACADM, HADHA. These enzymes are not only
important in the field of inborn errors of metabolism, but also in
metabolic syndrome, type 2-diabetes and compound toxicity.
3
ACADVL is a homodimer that carries out the first step toward esters
of long-chain and very long chain fatty acids such as palmitoyl-CoA,
mysritoyl-CoA and stearoyl-CoA. It can accommodate substrate acyl
chain lengths as long as 24 carbons, but shows little activity for
substrates of less than 12 carbons. Defects in ACADVL are the
cause of acyl-CoA dehydrogenase very long chain deficiency
(ACADVLD) [MIM:201475]. It is clinically heterogeneous, with three
major
phenotypes:
(1)cardiomyopathy
(2)a
hypoketotic
hypoglycemia and (3) rhabdomyolysis triggered by exercise or
fasting.
ACADM is a homotetramer and it also carries out the first step in the
pathway but, as opposed to ACADVL, it is specific for acyl chain
lengths of 4 to 16 carbons. Defects in ACADM are the cause of acylCoA
dehydrogenase
medium-chain
deficiency
(ACADMD)
[MIM:201450], the most common fatty acid oxidation disorder in
humans. This is an autosomal recessive disease which causes
fasting hypoglycemia, hepatic dysfunction, and encephalopathy,
often resulting in death in infancy.
The trifunctional enzyme (TP) is an octomer (4 alpha and 4 beta
subunits) that catalyzes three out of the four steps in beta oxidation
pathway with a specific affinity for long chain substrates. This kit
measures the alpha subunit of the trifunctional protein which
catalyzes the hydration of enoyl-CoA and the dehydrogenation of 3hydroxyacyl CoA compounds (steps 2 and 3). Defects in HADHA
4
are a cause of TP deficiency [MIM:609015], long-chain 3-hydroxylCoA dehydrogenase deficiency (LCHAD deficiency) [MIM:609016]
and maternal acute fatty liver of pregnancy (AFLP) [MIM:609016].
2. Assay Summary
Harvest cells as a single cell suspension.
Fix cells with 4% paraformaldehyde for 15 minutes and pellet
Permeabilize cells with 1X permeabilization buffer for 15 minutes
and pellet
Block cells with Blocking/Incubation buffer for 15 minutes and
pellet
Overlay 2X primary antibodies diluted in Blocking/Incubation
buffer; incubate for 1 hour and wash.
Add 1X secondary antibodies diluted in Blocking/Incubation
buffer, incubate for 1 hour, wash and read with flow cytometer
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3. Kit Contents
Item
Quantity
10X Phosphate Buffered Saline (PBS)
100 mL
100X Triton X-100 (10% solution)
1.25 mL
400X Tween – 20 (20% solution)
4 mL
Blocking Buffer
25 mL
100X ACADVL Primary antibody
0.12 mL
100X ACADM Primary Antibody
0.12 mL
100X HADHA Primary Antibody
0.12 mL
100X Normal Mouse IgG
0.12 mL
4. Storage and Handling
Spin down the contents of the antibody vials upon receipt of the kit.
Store all components upright at 4°C. This kit is stable for at least 6
months from receipt.
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5. Additional Materials Required

Flow cytometer.

Microfuge.

20% paraformaldehyde.

Goat
anti-mouse
detection
antibodies
labeled
with
fluorochrome(s) suitable for available flow cytometer (e.g.
Abcam, GAM IgG – H&L DyLight® 488 (ab96871)).
6. Preparation of Reagents
6.1 Prepare 1X PBS by diluting 100 mL of 10X PBS in 900 mL
Nanopure water or equivalent. Mix well. Store at room
temperature.
6.2 Immediately prior to use prepare sufficient 1X Wash Buffer
= 0.05% Tween-20, 1% Blocking buffer in PBS.
6.3 Immediately prior to use prepare enough 1X
Permeabilization Buffer = 0.1% Triton X-100 in PBS.
6.4 Immediately prior to use prepare 1X Incubation buffer =
10% blocking solution, 0.1% Triton X-100 in PBS. Any
excess should be stored at 4°C for no more than 24 hours.
7
7. Sample Preparation
7.1
Cell culture and treatment conditions are dictated by the
experiment at hand. As a general guideline, it is advisable
to analyze at least 10,000 events (cells) on the flow
cytometer per sample/data point. Therefore at least four
times that many cells should be collected per data point to
ensure sufficient material at the end of the staining.
7.2
For suspension cells, generate a single cell solution by
pipetting up and down.
7.3
For adherent cells, fully dissociate cells (e.g. trypsin) into
single cell suspension. Passaging the cell line the day
before the experiment onto a fresh cultured plate may help
improve single cell dissociation on the day of the
experiment.
7.4
Pellet cells at 350 – 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant. Note: It is helpful
during the fixation process to leave no more than 100µL of
complete media over the cell pellet.
7.5
Resuspend pellet into a suitable volume of PBS to a final
concentration of 2–5x106 cells per mL.
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8. Assay Procedure
8.1
Overlay 20% paraformaldehyde such that the final
concentration is 4%, gently mix by inverting the tube and
incubate at room temperature for 15 minutes.
8.2
Pellet cells at 350 - 500 x g for 5 minutes (depending on
cell
size)
and
decant
supernatant.
Note:
paraformaldehyde is toxic: handle with care and dispose of
according to local requirements.
8.3
Dislodge the pellet by gently tapping the bottom of the
tube and add 1X Permeabilization buffer to a final
concentration of 1–2x106 cells per mL.
Mix by gently
inverting the tube and incubate at room temperature for 15
minutes.
8.4
Pellet cells at 350 – 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.5
Dislodge the pellet by gently tapping the bottom of the
tube and add 50µL of 1X Blocking/Incubation buffer per
tube (final concentration is 2x104 cells per µL). Mix by
gently inverting the tube and incubate at room temperature
for 15 minutes.
9
8.6
Prepare 50µL per assay tube of 2X ACADVL Primary
Antibody Solution in 1X Blocking/Incubation buffer so that
it can be overlayed over the cell suspension for a final 1X
antibody solution.
8.7
Repeat previous step for ACADM and HADHA antibodies.
Note – All antibodies are mouse in origin and should not
be mixed.
8.8
Overlay primary antibodies over the cell suspension and
mix by gently inverting the tube.
Incubate at room
temperature for at least 1 hour.
8.9
Pellet cells at 350 - 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.10 Dislodge the pellet by gently tapping the bottom of the
tube and wash twice by centrifugation with 1mL of 1X
Wash per assay tube.
8.11 Prepare 100µL of Goat Anti-Mouse detection antibody
labeled with a fluorochrome in 1X Blocking/Incubation
buffer per assay tube.
8.12 After final wash, dislodge the pellet by gently tapping the
bottom of the tube and add 100µL of detector solution per
assay tube. Incubate for at least 1 hour at room
temperature in the dark.
10
8.13 Pellet cells at 350 – 500 x g for 5 minutes (depending on
cell size) and aspirate supernatant.
8.14 Dislodge the pellet by gently tapping the bottom of the
tube and wash twice by centrifugation with 1mL of 1X
Wash per assay tube.
8.15 After final wash, dislodge the pellet by gently tapping the
bottom of the tube and add 100µL of PBS to each assay
tube.
9. Data Analysis
Specific methods depend on the available flow cytometer.
It is
important to appropriately establish forward and side scatter gates to
exclude debris and cellular aggregates from analysis.
This kit
provides a Normal Mouse IgG as a negative antibody control and
signal from this antibody can be used to determine background
fluorescence.
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10.
Assay Performance and Specificity
All the antibodies used in this assay are IP positive and were
previously
validated
by
mass
spectrometry
of
the
immunoprecipitated protein (data not shown, but available upon
request).
Assay specificity was demonstrated by using primary
fibroblasts cell lines from patients with a well characterized
enzymatic deficiency in one of the FAO enzymes. Figure 1 shows
results after testing cell lines with well characterized mutations and
deficiencies
in
these
enzymes
(1)
ACADVL:p.[N122D],
(2)
ACADM:p[K304E and (3) HADHB:p[R61H];[R247H]. Although the
mutation was in the HADHB gene, levels of HADHA were found
depleted by flow cytometry, immunocytochemisty and western blot
perhaps due to post-transcriptional instability of the assembled
trifuntional protein (HADHA + HADHB).
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Levels of ACADM in
control and deficient cell line
un
ro
kg
de
C
fi c
on
ie
tr
d
nt
d
kg
B
D
A
H
LC
A
A
C
C
A
A
D
0
ro
fi c
de
M
ac
B
VL
D
2.010 4
un
ol
tr
C
kg
on
ro
fi c
de
nt
0
4.010 4
ol
5.010
4
6.010 4
ac
Mean Fluorescent Intensity
1.010 5
un
ie
tr
on
C
1.510 5
d
nt
0
2.010 5
ie
1.010 5
Levels of HADHA in
control and deficient cell line
8.010 4
B
2.010 5
2.510 5
ac
Mean Fluorescent Intensity
3.010 5
ol
Mean Fluorescent Intensity
Levels of ACADVL in
control and deficient cell line
4.010 5
Figure 1. Antibody specificity demonstrated by flow cytometry. Control
(blue) and deficient cell lines (red) were targeted with the FAO antibody
panel against ACADVL, ACADM and HADHA. Background fluorescence
was determined with the supplied negative control (black). After background
subtraction, the ACADVL deficient cell line shows a 93% reduction in the
level of the ACADVL protein, the ACADM deficient line shows a 57%
reduction in the level of the ACADM protein and the LCHAD deficient cell
line shows a 50% reduction of in the level of the HADHA protein.
Confidence
in
antibody
specificity
is
critical
to
Flow
data
interpretation. Therefore all antibodies in this kit were also tested for
specificity
by
fluorescence
immunocytochemistry
conditions used for the Flow assay.
under
the
Furthermore levels of the
proteins were independently validated by western blot in control and
deficient cell lines. The antibody against ACADVL is only ICC and IP
positive and therefore the levels of ACADVL in the deficient cell line
were
validated
demonstrating
with
the
Abcam
antibody
immunocytochemical
ab54698.
and
Examples
Western
blot
specificities of the primary antibodies used in this panel are shown in
Figures 2 and 3.
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Figure 2. Antibody specificity demonstrated by immunocytochemistry.
Cells were processes with the Flow cytometry protocol as explained above.
Panel A shows control fibroblasts and panel B shows deficient fibroblasts.
Left panel shows staining with anti-ACADVL ab, center panel with antiACADM ab and right panel with anti-HADHA ab.
14
Figure 3. Validation of antibodies and cell lines by WB. Western blot was
run on a 10-20% gradient acrylamide gel.
Samples were loaded at
40ug/lane for anti-ACADM and anti-HADHA antibodies and at 20ug/lane for
the anti-ACADVL antibody. Deficient samples used were the same as those
used for the flow cytometry assay.
15
11.
Troubleshooting
Problem
Low Signal
Cause
Solution
Signal not correctly
Check positive single color
compensated
control is set up correctly on
flow cytometer and
gated/compensated correctly
to capture all events.
Insufficient
Increase concentration of
secondary antibody
antibody.
Lasers not aligned
Run flow check beads and
adjust alignment if necessary
Second antibody is
Use secondary antibody
not compatible with
raised against mouse for this
primaries
kit.
16
Problem
High side
Cause
Solution
Cells lysed
Samples should be freshly
scatter
prepared. Do not vortex or
back-
shake the sample at any
ground
stage. Do not exceed 500 x g
for centrifugation.
Low event
rate
Bacterial
Ensure sample is not
contamination
contaminated.
Low number of cells
Run 1x106 cells/mL.
Cells clumped
Ensure a single cell
suspension. Sieve clumps
(30 µL nylon mesh).
High event
High number of
Dilute between 1x105 cells/mL
rate
cells/mL
and 1x106 cells/mL.
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