AMPK Methods and Protocols

3.3 Monocyte
Adhesion Assay

1. Grow endothelial cells to confluence on 24-well plates—4-
   wells per condition to give four technical replicates.


2. Grow U937 monocytes in T150 flasks, maintaining a density of
   1–2 105 cells/ml (seeNote 27).


3. On the day of the experiment, aspirate endothelial cell medium
   and replace with 0.5 ml/well of fresh medium.


4. Add pharmacological AMPK activator (A769662 at final con-
   centration of 100μM. 1μl of 50 mM stock/well (seeNote 6))
   or equivalent volume of DMSO to control wells (seeNote 7)
   and return to a CO 2 incubator at 37C.


5. After 30 min incubation with A769662, remove plate from the
   incubator and add pro-inflammatory cytokine (IL-1βat final
   concentration of 10 ng/ml—5μlof1μg/ml) to appropriate
   wells.


6. Return plate(s) to a CO 2 incubator at 37C and incubate for
   6h.


7. Wash U937 pro-monocytic cells into serum-free RPMI 1640
   medium and resuspend at a concentration of 2 105 U937
   cells/ml. Do this half an hour beforestep 8.


8. Aspirate medium from endothelial cells and wash monolayers
   three times with 0.5 ml/well of serum-free RPMI 1640 prior
   to overlay with 1 105 U937 cells/well (0.5 ml per well of
   24-well plate) (seeNote 28).


9. Allow cells to adhere for 1 h at 37CinaCO 2 incubator,
   aspirate the medium, and wash monolayers three times with
   1 ml/well serum-free DMEM to remove any nonadherent
   U937 cells (seeNote 29).


10. Fix cells for 25 min at room temperature in PBS-sucrose-PFA
    (400μl/well) (seeNote 30).


11. Aspirate PBS-sucrose-PFA and quench remaining PFA by
    washing each well twice with PBS-glycine (0.5 ml/well), fol-
    lowed by two washes with PBS (0.5 ml/well).


12. Count the number of adhered U937 cells per field of confluent
    ECs (at least two fields per well) on a light microscope with a
    5 objective. Plates can be stored at 4C prior to analysis, if
    preferred.

4 Notes

1. Add dilute NaOH dropwise when adjusting the pH of the
   working solution, as rapid changes in pH can cause the calcium
   to precipitate. If this happens, the buffer will become cloudy
   and should be discarded.

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