Fig. 3: Overview of current flow cytometry techniques available to probe cellular metabolism.
From: Approaches to investigate tissue-resident innate lymphocytes metabolism at the single-cell level
A Glutamine (Q) uptake assay with single-cell resolution (QUAS-R); bioorthogonal amino acids l-azidohomoalanine (AHA) and l-homopropargylglycine (HPG) are used to quantify amino acid transport through Slc1a5. B Kynurenine is a naturally fluorescence cargo for Large neutral amino acid (LAT1-4) transporters and is used to measure amino acid uptake through these transporters. In many lymphocytes, LAT1 (Slc7a5/Slc3a2 heterodimer) is the only LAT isoform expressed and so kynurenine uptake quantifies Slc7a5 mediated amino acid uptake. C SCENITH estimates single-cell energetic metabolism by profiling translation inhibition. Puromycin incorporation is used to quantify protein translation rates and to probe energy metabolism. D Iron–transferrin uptake is measured using a fluorescently tagged transferrin molecule. E Mitochondrial dyes can be used to measure mitochondrial mass, mitochondrial ROS and polarisation across the mitochondrial inner membrane. F METFLOW is a high-parameter flow cytometry method utilising antibodies against metabolic proteins; that probes the capacity of a cell to engage different metabolic pathways. Essential to the accurate interpretation of these assays are robust controls; for instance, competition controls are imperative for QUAS-R and Slc7a5 uptake analysis. Protocol A is compatible with protocol B, and protocol C. Further effort is being made to integrate the remaining parameters. This figure was created in BioRender. Finlay (2024) https://BioRender.com/d06l575.
