Intracellular Signaling: Where Full Kinetics Data Excel - New Publication

A new publication describes really inspiring results: The authors report how they used xCELLigence cell assays to remove an ambiguity due to contradictory previous findings regarding PKC receptors - based on the instrument's ability of continuous long-term measurements with full-kinetics read-outs.


Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165

Deissler H.L. · Lang G.K. · Lang G.E.. Cell Physiol Biochem 2017;42:1493–1513. https://doi.org/10.1159/000479213

Enhanced permeability of retinal endothelial cells (RECs) is a hallmark of macular edema and is induced by growth factor VEGF-A165. Using xCELLigence, the authors investigated multiple inhibitors of intracellular kinases blocking harmful effects of VEGF-A165 on iBREC (immortalized bovine retinal endothelial cells).

xCELLigence enabled continuous measurements of cell permeability over several days without interfering otherwise, allowing to monitor small or transient effects - in contrast to conventional TER (trans endothelial resistance) measurements.

Dr. Heidrun Deissler, University of Ulm, Germany:

"What really impressed us with xCELLigence technology was the fact that effects observed by others in capillaries in vivo, were similarly recorded by xCELLigence: After addition of VEGF-A, the CI (cell index), a measure of cell barrier integrity, dropped sharply, recovered quickly before values started to decrease continuously for at least 3 days.

In this paper we were able to observe a transient and reversible destabilization of the iBREC barrier by a MEK inhibitor. Interestingly, this might provide a rationale for the appearance of subretinal fluid in patients receiving anti-cancer treatment with MEK inhibitors.

Regarding PKC (protein kinase C) inhibitor GF109203X, xCELLigence continuous CI measurements allowed us to demonstrate the complete inhibition of the early transient barrier disturbance induced by VEGF-A whereas the inhibitor only partly blocked the later sustained response - all measurements were possible in one and the same iBREC culture!

This allowed us to clearly distinguish phases of VEGF-A action and clarified previous findings reporting contradictory observations related to PKC inhibitors.

In a nutshell, due to its continuous and sensitive measuring principle, xCELLigence cell-based assays provided us with new and refined insights into intracellular signaling and helped to clarify previous inconsistent observations. xCELLigence established itself as an indispensable tool in our lab.”

 

Figure: Effects researchers might miss solely relying on endpoint-assays:  Inhibitor of PKC GF109203X prevented only early transient and but not late sustained response of IBREC to VEGF-A (green line). Red line: cell barrier integrity drops sharply after addition of VEGF-A, recovers and continues to decline over several days. From the publication, © the authors.

 

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