Table 5.

Application of chemical-based and protein-based fluorescent sensors to study free zinc in enterocytes.

Cell Model	Sensor	Incubation Parameter	Main Outcome	Reference
HT-29 Cultivation time: -proliferating cells: 24 h -resting cells: 48 h (serum depleted) -differentiated cells: 6 days (first 72 h with sodium butyrate); 2D	FluoZin-3 (Kd = 8.9 nM) Newport Green (Kd = 30 µM) (low molecular weight sensors)	-sensor pre-incubation: 0.3–5 µM FluoZin-3 or 5µM Newport Green for 30 min in DPBS -spectrofluorometer	-free zinc in HT-29 0.6–1.2 nM for proliferating, resting or differentiated cells -a surplus of zinc-binding proteins buffer the intracellular free zinc concentration and guarantee a stable zinc homeostasis	[340]
Caco-2/TC7 Cultivation time: 15-17 days; 2D	FluoZin-3 (Kd = 15 nM) Zinquin (low molecular weight sensors)	-sensor pre-incubation: 1 µM FluoZin-3; 25 µM Zinquin -samples were fixed with paraformaldehyde prior staining -fluorescence microscope	-both sensors accumulate in vesicle-like structures -imaging of free zinc distribution and tight junction formation in enterocytes	[339]
HT-29 Cultivation time: n.a.; 2D	Newport Green (low molecular weight sensor)	-sensor pre-incubation: 5 µM Newport Green for 30 min in assay buffer a -fluorescence microplate reader	-increase of intracellular free zinc levels after zinc treatment are lower than changes in total cellular	[341]
Caco-2 Cultivation time: 17 days; 2D	FluoZin-3 (Kd = 15 nM) (LMW sensor)	-sensor pre-incubation: 1 µM FluoZin-3 for 1 h in OptiMEM, -fluorescence microscope and microplate reader	-sensor accumulates in vesicles -basal free zinc decreases after treatment with hepcidin	[156]
Caco-2 Cultivation time: 10 days; 2D	Zinypr-1 (Kd = 0.7 nM) (low molecular weight sensor)	-sensor pre-incubation: 50 µM Zinpyr-1 for 1 h in PBS -fluorescence microscope	-zinc uptake from different zinc-complexes with amino acids	[96]
Caco-2-eCalwy Cultivation time: resting state; 2D	eCalwy-5 (Kd = 1.85 nM) (Genetically encoded protein-based sensor)	FRET and FLIM-FRET measurements using low molecular weight (LSM) in assay buffer b	-eCalwy protein is mainly localized in the cytoplasm of the Caco-2-eCalwy clone -cytoplasmic free zinc was estimated to be around ~2 nM	[101]
Caco-2 Cultivation time: 21 days; 2D	Zinpyr-1 (Kd = 0.7 nM) (low molecular weight sensor)	-sensor pre-incubation: 2.5 µM Zinpyr-1 for 30 min in assay buffer + 0.3% BSA -fluorescence microplate reader	-sensor accumulates in cellular vesicles -basal free zinc was estimated to be ~0.2 nM	[102]
Caco-2 Cultivation time: 21 dHT-29, HT-29-MTX Cultivation time:7 days; 2D	Zinypr-1 (Kd = 0.7 nM) (LMW sensor)	-sensor pre-incubation: 2.5 µM Zinpyr-1 for 30 min in assay buffer + 30% BSA -fluorescence microplate reader	-sensor accumulates in cellular vesicles (HT-29, HT-29-MTX, Caco-2) -basal free zinc was estimated to be ~0.5 nM in HT-29-MTX, 0.8 nM for HT-29	[103]

2D, two-dimensional. BSA, bovine serum albumin. DMEM, Dulbecco’s Modified Eagles Medium. DPBS, Dulbecco’s phosphate buffered saline. FCS, fetal calf serum. FLIM, fluorescence lifetime imaging microscopy. FRET, Förster resonance energy transfer. HBSS, Hank’s Balanced Salt Solution. HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. LSM, laser scanning microscope. n.a., not available. PBS, phosphate buffered saline. ^a 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl₂, 20 mM Hepes, 15 mM glucose, 1.8 mM CaCl₂, 10 mM NaOH, pH 7.4. ^b 120 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM NaH₂PO₄, 10 mM HEPES, pH 7.35.