Deciphering the Intracellular Signaling of Erythropoietin in Neuronal Cells - Tissue-Protective Cytokines: Methods and Protocols - page 174

2.4 Akt Kinase
Activity Assay

1. AKT Kinase Assay Kit (Nonradioactive; Cell Signaling,
#9840).
2. Assay Lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X, 2.5 mM
sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM
Na 3 VO 4 , 1 μ g/ml leupeptin, 1 mM phenylmethylsulfonyl

fluoride).
3. Kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM b-glycero-
phosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na 3 VO 4 ,
10 mM MgCl 2 ).
4. ATP (included in the Akt Kinase assay kit).
5. GSK-3 Fusion Protein (included in the Akt Kinase assay kit).
6. LumiGLO ® Substrate (Cell Signaling, #7003).

Materials for primary rat neuronal cultures:

2.5 Primary Mixed
Cultures

1. Poly- L -lysine.
2. Borate Buffer (pH 8.0).
3. Sprague Dawley Rat, pregnant (Charles Rivers).
4. Culture plates.
5. Glass coverslips (Fisher Scientific, Fisherbrand Coverglasses,
Circles No. 2—0.17-0.25 mm thick; Size: 22 mm; Catalog
no. 12-546-1) (see Note 2).
6. 70% nitric acid.
7. 100% ethanol.
8. Two #5 tweezers with sharp and pointy tips.
9. 1 retina scissor.
10. Hank's balanced salt solution (pH 7.2).
11. 0.25% Trypsin-EDTA.
12. Dulbecco's Modified Eagle Medium (DMEM).
13. F-12 Nutrient Mixture.
14. Heat inactivated bovine serum.
15. L -Glutamine.
16. HEPES buffer.
17. Penicillin.
18. Streptomycin.
19. Pasteur pipets with fire polished tips (final opening diameter at
tip >300 m).

20. Cell strainer, 40

μ

μ m (BD Falcon, 352340).