Alfredo Caro Maldonado

Product Specialist of Seahorse XF Consumables, Spain, Portugal, Agilent Technologies

The next generation of immune cell therapies require a new generation of analysis tools

A safe, potent, and persistent immune cell product depends on developers mustering the full repertoire of immune cell function; activation, proliferation, cell fate, cytotoxic killing, immunomodulation, and memory – all of which must be accomplished in the ever-changing, suppressive, and toxic tumor microenvironment. Agilent is dedicated to supporting these next-generation therapies, providing key technologies to enable high-efficiency gene editing and assessment of real-time cell function, phenotype, and fate.

Agilent Seahorse XF technology is the gold standard method to measure cell bioenergetic metabolism in life cells in real time. Bioenergetic metabolic measurements in immune cells can provide insightful information about cell function and modulation of metabolic pathways can be utilized to re-direct cell fate. In this workshop we will discuss how XF Seahorse assays have been successfully applied to address the challenges of cell engineering and manufactory process during the design and development of new immunotherapeutics.

• • Ponente: Alfredo Caro Maldonado
  • Afiliación: Product Specialist of Seahorse XF Consumables, Spain, Portugal, Agilent Technologies
  • email: alfredo.caro-maldonado@agilent.com

Alfredo Caro Maldonado

Product Specialist of Seahorse XF Consumables, Spain, Portugal, Agilent Technologies

Agilent Seahorse XF Analyzers have been the cornerstone of metabolic studies in live cells for over a decade. There are a variety of assay approaches we can use to examine metabolic phenotypes and changes in the cell. For example, we can evaluate glycolytic or mitochondrial function, substrate oxidation, or the production rate of ATP from each of these pathways. However, determining more mechanistic aspects of mitochondrial function historically has involved analyzing isolated mitochondria or detergent permeabilized cells in a single chamber using an oxygen electrode. These techniques allow the researcher direct access to the mitochondrion. This is necessary, in part, because the plasma membrane inhibits certain molecules or substrates of interest, such as succinate, from entering the cell in sufficient concentrations for study.

In this talk there will be:

• A brief discussion on isolating mitochondria from tissues and the assays one can use, using much less mitochondrial mass for the same amount of data as other. This was covered, in part, in a previous webinar presented by Manish Bharadawaj of Agilent. We will discuss coupling and electron flow assays and look at published data.
• An introduction and discussion on permeabilizing cells with the Agilent XF Plasma Membrane Permeabilization (PMP) reagent. This is a proprietary, protein-based reagent that requires minimal or no optimization. PMP is non-destructive to the mitochondrial membrane, an issue when using detergents, and assay preparation is much quicker than isolating mitochondria. PMP can be used with adherent cells, which requires much less biomass than other techniques. This allows one to do more varied experiments with genetically modified cell lines. We will also examine published data where PMP has been used.