Table 1 Overview of the six IFC assays quantifying six different aspects of cellular health. All assays were validated using compounds known to cause specific changes in the targeted pathway, serving as positive controls. Next, all assays were validated using patient cells, with diseases known to cause specific changes in the pathway of interest (positive patient control)
Primary assay | Marker | Molecular compound | Positive patient control |
|---|---|---|---|
Mitochondrial mass | NAO | Valproic acid [43] | Polymerase-y (POLG) pathogenic variants [44] |
Membrane potential | TMRM | FCCP [45] | Mitochondrial disease patients with mt-TL1 or NDUSF4 pathogenic variants [46, 47] |
Autophagy | LC-3 | Bafilomycin and starvation [48] | EPG5 pathogenic variants [49] |
Lysosomal accumulation | LAMP-1 | NA | CLN3 pathogenic variants [28] |
ER stress | ATF6 | Brefeldin A [50] | PMM2 pathogenic variants [51] |
Golgi fragmentation | GM130 | Nocodazole [52] | TRAPPC2L pathogenic variants [53] |
NF-κβ translocation | p65 | TNF-α [54] | NFKB1 pathogenic variants [55] |