Biology Reference
In-Depth Information
membrane. Following a wash to remove unbound material,
Streptavidin-HRP, and chemiluminescent detection reagents are
added sequentially. The light is produced at each spot in propor-
tion to the amount of cytokine bound and can be quantifi ed by
exposing membranes to the fi lm with subsequent quantifi cation of
the size and density of the light-producing areas by histogram tool
of imaging software or by other available techniques. The detailed
description of kit components, methods, standard handling and
optimization procedures are provided within product description
to ProteomeProfi ler™ Antibody Array Kit (R&D Systems). Here
we describe our optimized methods for using this kit for the quali-
tative and quantitative analyses of cytokines and chemokines in
mouse spleen and plasma after mouse infection with Ad.
1. Mice are injected with Ad via a tail vein infusion and sacrifi ced
by cervical dislocation to harvest tissues for subsequent analy-
ses 1 h after virus administration (
see
Note 1
).
2. Spleen is recovered, cleaned from any fat-containing surround-
ing tissues, and 50 mg of total splenic tissue is quickly placed
into 2 mL of lysis buffer and homogenized with a tissue
homogenizer.
3. Add Triton X-100 to a fi nal concentration of 1 %. Freeze sam-
ples at
3.1 Analysis of the
Relative Amounts
of Pro-Infl ammatory
Cytokines and
Chemokines in Mouse
Spleen After
Intravenous Ad
Injection
−70 °C, thaw, and centrifuge at 10,000 ×
g
for 5 min
to remove cellular debris.
4. Proceed to preparation of an antibody array membrane by
pipetting 2.0 mL of Array Buffer 6 into each well of the 4-Well
Multi-dish supplied within the ProteomeProfi ler™ kit. Array
Buffer 6 serves as a block buffer.
5. Using fl at-tip tweezers, remove antibody array membrane from
between the protective sheets and place in a well of the 4-Well
Multi-dish. The number on the membrane should be facing
upward.
6. Incubate for 1 h on a rocking platform shaker. Orient the tray
so that each membrane rocks end to end in its well.
7. While the membranes are blocking, prepare samples by adding
up to 1 mL of each sample to 1 mL of Array Buffer 4 in sepa-
rate tubes. Adjust to a fi nal volume of 2 mL with Array Buffer
6 as necessary.
8. Add 15
≤
L of reconstituted Mouse Cytokine Array Panel A
Detection Antibody Cocktail to each prepared sample. Mix
and incubate at room temperature for 1 h.
9. Aspirate Array Buffer 6 from the wells of the 4-Well Multi-dish
and add sample/antibody mixtures prepared in
steps 7
and
8
.
Cover the 4-Well Multi-dish with the lid.
10. Incubate overnight at 2-8 °C on a rocking platform shaker.
μ
