cells are not exposed directly to them. In that case, after wash-
ing, the conditioned medium can be collected over an hour and
used to examine cell function, as we have previously described
for U937 cell migration toward conditioned medium [9].- For HUVECs we find a dilution of 1:40 in calibrator diluent
RD5L is necessary. - Make sure to aspirate the samples entirely using a water/pump-
powered aspirator if available. The first two washes can be
discarded simply by inverting the plate over a tub or sink and
giving a shake. The final wash should be aspirated entirely and
the plate blotted against paper towels. - This should be added to wells in the same order as the substrate
solution was added. The color should change from blue to
yellow upon addition of the “stop solution.” If the color
appears greenish in some wells, use a 200μl pipette to gently
mix. Be sure to change the tip if mixing multiple wells. - A wavelength of 485 nm is also adequate if 450 nm is not
possible. - This is to account for optical imperfections in the plate.
- U937 cells are grown in suspension, so agitate flask while
dispensing cells to ensure they are thoroughly mixed. - Washing ensures U937 cells are not exposed to test substances,
ensuring observed effects are mediated by ECs and not due to
direct actions of pro-inflammatory stimuli or AMPK activators
on U937 function. - Wash monolayer gently—use a larger pipette for less force, e.g.,
a 25 ml pipette—and trickle down the side of the well. - The presence of sucrose is essential to prevent osmotic lysis of
monocytes.
References
- Nunemaker CS (2016) Considerations for
defining cytokine dose, duration, and milieu
that are appropriate for modeling chronic
low-grade inflammation in type 2 diabetes. J
Diabetes Res 2016:2846570 - Sansbury BE, Spite M (2016) Resolution of
acute inflammation and the role of resolvins in
immunity, thrombosis, and vascular biology.
Circ Res 119:113–120 - Libby P, Nahrendorf M, Swirski FK (2016)
Leukocytes link local and systemic inflamma-
tion in ischemic cardiovascular disease: an
expanded “cardiovascular continuum”. J Am
Coll Cardiol 67:1091–1103 - Musolino C, Allegra A, Pioggia G, Gangemi S
(2017) Immature myeloid-derived suppressor
cells: a bridge between inflammation and can-
cer (Review). Oncol Rep 37:671–683- Antonioli L, Colucci R, Pellegrini C et al
(2016) The AMPK enzyme-complex: from
the regulation of cellular energy homeostasis
to a possible new molecular target in the man-
agement of chronic inflammatory disorders.
Expert Opin Ther Targets 20:179–191 - Steinberg GR, Schertzer JD (2014) AMPK
promotes macrophage fatty acid oxidative
metabolism to mitigate inflammation: implica-
tions for diabetes and cardiovascular disease.
Immunol Cell Biol 92:340–345 - Salt IP, Palmer TM (2012) Exploiting the anti-
inflammatory effects of AMP-activated protein
318 Sarah J. Mancini and Ian P. Salt