Table 1.

Tools for 96-well-plate-based immunometabolic profiling

Methods	Assay principle	Advantages/disadvantages	Equipment
extracellular glucose/lactate assay	estimate of glycolysis based on cumulative glucose consumption/lactate secretion	+ simple, fast, cheap	absorbance (glucose) or fluorescent (lactate) plate reader
		− limited insight
		− bulk analysis
NO/arginase assay	estimates L-arginine metabolism via iNOS/arginase	+ simple, fast, cheap	absorbance plate reader
		− not all species/cell types
		− bulk analysis
extracellular flux analysis	measures ECAR and OCR as proxies of glycolysis and mitochondrial OXPHOS, respectively	+ parallel readouts of glycolysis and OXPHOS	Seahorse XF analyzer
		+ adaptable injections and protocol
		− dedicated (costly) instrument/consumables
		− normalization needed
		− bulk analysis, require cell purification/sorting
SCENITH	estimates metabolic capacities and dependencies by measuring changes in the level of protein synthesis	+ suitable for rare cells and complex samples	flow cytometer
		+ compatible with complex immune phenotyping
		− assay principle requires active protein synthesis
		− subset analysis rather than single cell for calculated parameters
2-NBDG and BODIPY C16 uptake	fluorescent glucose analog and fluorescent fatty acid to estimate glucose or fatty acid uptake, respectively	+ simple, fast, single cell	flow cytometer/multi-mode fluorescence imager/microscope
		− need to be validated with complementary readouts and appropriate controls to ensure correct interpretation
MitoTracker Green and TMRM (TMRE)	fluorescent mitochondrial mass and mitochondrial membrane potential indicator, respectively	+ simple, fast, single cell	flow cytometer/multi-mode fluorescence imager/microscope
		− need to be validated with complementary readouts and appropriate controls to ensure correct interpretation