Category: Assay Development and Screening

1323-D - Implementation of acoustic dispensing for kinetic monitoring of glycolysis and glutaminolysis.

Tuesday, February 6, 2018
5:00 PM - 6:00 PM

Glycolysis and glutaminolysis are two major energy metabolism pathways required for rapid cell proliferation.  In the cancer microenvironment, cancer cells and activated T lymphocytes exhibit similar metabolic profiles as they compete for the same nutrients.  Understanding and targeting these metabolic pathways, therefore, can provide valuable information on the function of those cells and lead to more effective cancer treatment strategies that avoid impairing the immune system.  Contamination and carryover-free acoustic liquid handling technology has been widely implemented into high-throughput applications, including nanoliter volume dispensing of large compound libraries.  Here we evaluated the novel application of small volume acoustic sampling of cell culture medium from cells cultured in 384-well plates.  Normal usage of these high sensitivity metabolism assays requires sample pre-dilution.  In contrast, use of the Echo® Liquid Handler eliminates this pre-dilution step as it is able to dynamically adjust to differing fluid properties and is able to transfer from any well to any well.  This type of rapid and small volume sampling has minimal impact on the cell growth conditions, allowing for easy and fast repetitive cell metabolism monitoring.  Culture medium samples were collected at different time points into 1536-well plates and were analyzed using bioluminescent metabolite detection assays.  Glycolysis and glutaminolysis were monitored by measuring glucose and glutamine consumption and lactate and glutamate secretion in A549 cancer cells.  We showed that the changes in metabolite levels were time and cell number dependent.  Exposure to metformin, a potent antihyperglycemic agent and an inhibitor of the mitochondrial respiratory-chain complex 1, showed dose-dependent changes in metabolite levels.  By integrating the newly developed bioluminescent Lactate dehydrogenase (LDH) assay (sensitive enough to measure LDH levels in < 10 cells) into a workflow, we were able to normalize changes in cell metabolism with cell number.  Taken together, this data indicate that by combining small volume acoustic dispensing with the sensitivity and robustness of bioluminescent detection, we have developed a new approach for studying key cellular energy metabolism pathways that is well suited for high-throughput applications.

GEDIMINAS VIDUGIRIS

Senior Automation Scientist
Promega Corporation
Madison, WI

Gediminas Vidugiris is an Senior Automation Scientist at Promega Corp. Madison, WI.
He received his PhD in Biochemistry at Institute of Biochemistry, Lithuania Academy of Science, Vilnius, Lithuania. Dr. Vidugiris has extensive multidisciplinary research and development experience in systems automation, screening, assay development, biochemistry, biophysics, spectroscopy and electrochemistry.