AMPK Methods and Protocols

3.2 Preparation
of Conditioned
Medium from
Endothelial Cells
for the Analysis
of Cytokine
and Chemokine
Secretion

1. Grow human endothelial cells to confluence in 6-well plates—
   1-well per condition.


2. On the day of the experiment, wash cells into serum-free
   Medium 199 (1 ml/well) (seeNote 3).


3. Add pharmacological AMPK activator (A769662 at final con-
   centration of 100μM. 1μl of 100 mM stock/well (seeNote 6))
   or equivalent volume of DMSO to control wells (seeNote 7).


4. Return plate(s) to 37C incubator in atmosphere of CO 2.


5. After 30 min incubation with A769662, remove plate(s) from
   the incubator and add pro-inflammatory cytokine (IL-1βat
   final concentration of 10 ng/ml—10μl per well of 1μg/ml
   stock) to appropriate wells.


6. Return plate(s) to incubator and incubate for 6 h.


7. Remove plate(s) from the incubator and immediately transfer
   culture medium to 1.5 ml centrifuge tubes (seeNote 21).


8. Centrifuge conditioned medium at 3130gfor 20 min at 4C
   to pellet any cell debris. Transfer samples to clean 1.5 ml cen-
   trifuge tubes and store at 20 C.


9. Analyze MCP-1 levels by ELISA. We have obtained good
   results using the R&D Quantikine kits for MCP-1 or IL-6.
   We carried out the procedure as follows.


10. Optimize the dilution of conditioned medium required to
    achieve values within the linear range of the standard curve
    provided in the kit (seeNote 22).


11. Add 200μl of standard or sample (diluted as necessary) per
    well in duplicate and incubate for 2 h at room temperature on a
    vibrating platform at 200 rpm.


12. Aspirate each well and wash (400μl/well) three times (see
    Note 23).


13. Add 200μl of MCP-1 conjugate to each well and incubate at
    room temperature for 1 h on a vibrating platform as described
    instep 11.


14. Repeat the aspiration and wash as instep 12.


15. Add 200μl of “substrate solution” mix to each well. Incubate
    for 30 min at room temperature (not on shaking platform).
    Protect plate from light.


16. Add 50μl of “stop solution” to each well (seeNote 24).


17. Assess the optical density of each well using a microplate reader
    set to 450 nm (seeNote 25) with wavelength correction set to
    540 or 570 nm (if available). If wavelength correction is not
    available, subtract values obtained at 540/570 nm away from
    those obtained at 450 nm (seeNote 26).

314 Sarah J. Mancini and Ian P. Salt