Biology Reference
In-Depth Information
chemiluminescent image on the fi lm to a transparency overlay
template, provided with ProteomeProfi ler™ antibody array kit,
that defi nes the location of capture antibody spots on the
membrane for each individual cytokine and chemokine.
3.2 Analysis of the
Relative Amounts of
Pro- Infl ammatory
Cytokines and
Chemokines in Mouse
Plasma After
Intravenous Ad
Injection
1. Mice are injected with Ad via a tail vein infusion and sacrifi ced
by cervical dislocation to collect plasma for subsequent analy-
ses 1 h after virus administration (
see
Note 1
).
2. Use 20 mM EDTA in PBS (without calcium and magnesium)
or heparin as anticoagulant. Mix blood collected directly from
the heart or from vena cava well with EDTA (2 mM fi nal con-
centration) or heparin solution (10 U/mL fi nal concentration)
and place on ice while all samples are collected.
3. Centrifuge blood in a table-top microcentrifuge at 1,000 ×
g
for 5 min at +4 °C.
4. Carefully collect plasma atop of the pelleted blood cells into a
fresh tube. Avoid any cellular material.
5. Mix 100 mL of fresh plasma to a mixture of 1 mL of Array
Buffer 4 and 0.9 mL of Array Buffer 6.
6. Follow preparation of antibody array membranes, all solutions,
incubation times with buffers and all washing procedures as
described in Subheading
3.1
from
steps 4
-
25
.
Although simultaneous detection of 40 different mouse cytokines
and chemokines in mouse samples with ProteomeProfi ler™ anti-
body arrays provides highly reliable data in a quick and cost-
effective manner, the manufacturer (R&D Systems, Inc.) did not
aim this method to be used for quantitative detection of pro-
infl ammatory mediators. We adjusted and optimized antibody
array methodology with using these same reagents and kits for
quantitative detection of certain cytokines and chemokines, which
were prominently activated after mouse injection with Ad [
6
,
13
].
The basis for quantitative evaluation of specifi c cytokines and che-
mokines of interest in mouse samples was the standardization of
experimental conditions for incubation of the antibody array mem-
brane with negative control tissue samples spiked with different
known concentrations of purifi ed cytokine analyte and plotting
calibration curve that depicts the proportional relationship between
the amount of the cytokine added to a negative control sample and
the histogram signal on the fi lm, detected after its exposure to the
membrane for an exactly controlled periods of time.
3.3 Quantitative
Analysis of Specifi c
Infl ammatory
Cytokines and
Chemokines in Mouse
Samples After Ad
Infection Using
Antibody Arrays
1. Harvest spleen from mouse injected with PBS (instead of Ad)
1 h after PBS administration (
see
Note 1
).
2. Mix homogenized spleen sample with serial dilutions of specifi c
cytokines and chemokines, such as IL-1a, IL-1b, IL-6, KC, MIP-
2, or MCP-1, in a range of concentrations from 10 to 10 ng.
