Biology Reference
In-Depth Information
10. Plastic transparent sheet protector (trimmed to 10 × 12 cm and
open on three sides).
11. Plastic wrap.
12. Absorbent lab wipes (KimWipes
®
or equivalent).
13. Paper towels.
14. Autoradiography cassette.
15. Film developer.
16. X-ray fi lm (Kodak
®
BioMax™ Light-1, Catalog # 1788207) or
equivalent.
17. Flat-tipped tweezers.
18. Flatbed scanner with transparency adapter capable of transmis-
sion mode.
19. Computer capable of running image analysis software and
Microsoft
®
Excel.
20. Lysis buffer: PBS with inhibitors (pH 7.4), 1 % NP-40, 0.5 %
sodium deoxycholate, 1 mM Na
3
VO
4
, 0.1 % SDS, 1 mM
EDTA, 1 mM EGTA, 20 mM NaF, 1 mM PMSF.
21. ProteomeProfi ler™ antibody array kit, Mouse Cytokine Array
Panel A (Catalog # ARY006; R&D Systems, Minneapolis
MN).
22. Tissue homogenizer (OMNI-TH, TH115 or equivalent).
1. Phosphate-Buffered Saline (PBS), 20 mM NaCl, 2.68 mM
KCl, 10 mM Na
2
PO
4
, and 1.76 mM KH
2
PO
4
(pH 7.4).
2. Recombinant purifi ed mouse cytokines IL-1
2.2 Quantitative
Cytokine and
Chemokine Analysis
α
(Peprotech,
(Peprotech, Catalog #211-11B),
IL-6 (Peprotech, Catalog #216-16).
3. Recombinant purifi ed mouse chemokines MCP1 (Peprotech,
Catalog # 250-10), KC (Peprotech, Catalog # 250-11), MIP2
(Peprotech, Catalog #250-15).
Catalog #211-11A), IL-1
β
3
Methods
The main principle of simultaneous detection of numerous cyto-
kines and chemokines in tissues or body fl uids relies on interaction
of these cytokines and chemokines with specifi c capture antibodies
that have been spotted in duplicate on nitrocellulose membranes.
Tissue homogenates, serum, or plasma samples are diluted and
mixed with a cocktail of biotinylated detection antibodies.
The sample/antibody mixture is then incubated with the Mouse
Cytokine Array membrane supplied with ProteomeProfi ler™ anti-
body array kit. Any cytokine/detection antibody complex present
is bound by its cognate immobilized capture antibody on the
