TABLE 6.
Studies on Giardia EVs.
| Reference | Cell amounts | Vesiculation general conditions | Cell viability at time of EV harvest | Pre‐isolation/cell debris remotion | EV isolation and/or purification | EV characterization | EVs yield per batch (size and concentration) | In vitro/in vivo experiments using EVs | Markers |
|---|---|---|---|---|---|---|---|---|---|
| (Evans‐Osses et al., 2017) | 1 × 106 trophozoites/mL | 1 h‐incubation at 37°C in serum free YISa +1 mM CaCl2 (1 mL) | NR | C (2500 × g, 5 min; 4000 × g, 30 min) | UC (100,000 × g, 1.5 h) | NTA, TEM, FC |
∼201 nm (NTA) 5.77 × 108/mL |
PA Attachment to Caco‐2 cells Immature Dendritic cells (iDCs) stimulation Uptake by iDCs Lipid raft dependence assays |
NR |
| (Ma'ayeh et al., 2017) | NR |
2 h‐incubation and 6 h at 37°C in RPMI (50 mL) |
∼98% | C (930 × g, 10 min) and filtration (0.22 um), protease inhibitor treatment |
Concentration with Amicon Ultra 3 kDa cutoff AlbuminOUT kit (serum residue elimination) ExoQuick TC Kit |
TEM |
NR (Focus on whole secretory products, not in EVs) |
Uptake by intestinal epithelial cells in vitro | NR |
| (Moyano et al., 2019) | 14 × 107 trophozoites in total |
4 h‐incubation at 37°C in Bovine bile free/serum free TYI‐S‐33b |
NR | C (1455 × g, 15 min) and filtration (0.11 mm) |
UC (100,000 × g, 3.3 h) SG (1.03−1.25 g/cm3) separation UC of SG fractions (200,000 × g, 1 h) |
DLS, TEM |
50‐100 nm (DLS) 80 nm in a SG enriched fraction (TEM) Concentration NR |
Biogenesis investigation (detection of ESCRT‐ associated proteins by CLSM and TEM) |
actin, tubulin, g14‐3‐3, gQa1, PDI2 |
| (Gavinho et al., 2020) | 1 × 106 trophozoites/mL |
1 h‐incubation at 37°C in serum free TYIS‐33b +1 mM CaCl2 (1 mL) |
NR | C (600 × g, 5 min; 4000 × g, 30 min) |
C (15,000 × g, 1 h)—Large extracellular vesicles (LEV) UC (100,000 × g, 4 h)—Small extracellular vesicles (SEV) |
Protein Micro BCA assay, NTA |
187.6 nm—LEV 67.7 nm—SEV ∼1 × 109/mL—LEV ∼1 × 108/mL‐ SEV |
PA EVs inhibition assays (Cl‐amine and Cannabidiol) Effect of EVs on Caco‐2 cells/parasite adherence Uptake by Caco‐2 cells |
NR |
| (Zhao et al., 2021) | 1 × 106 trophozoites/mL |
12 h‐incubation at 37°C in TYI‐S‐33b with exosome‐depleted serum (1 mL) |
NR |
C (2000 × g, 10 min; 10,000 × g 45 min) and filtration (0.22 mm) |
UC (100,000 × g, 1 h) | Protein Micro BCA assay, NTA, TEM |
143.5 nm (NTA) 4,7 × 1010/mL |
PA Uptake by murine peritoneal macrophages (MPM) Stimulation of MPM (qPCR and ELISA to evaluate cytokines transcription and secretion) |
NR |
| (Grajeda et al., 2022) | 1 × 107 trophozoites in total |
3 h‐incubation at 37°C in PBS with 5 mM L‐cysteine, 5 mM glucose, and 1 mM CaCl2, pH 7.1 |
>99% | C (600 × g, 5 min; 4000 × g, 30 min) |
C (15,000 × g, 1 h)—Large vesicles UC (100,000 × g, 4 h)—Small vesicles |
NTA, TEM |
100‐400 nm—large vesicles <100 nm—small vesicles Concentration NR |
PA Treatment with giardial lipid raft (gLR) disruptors |
NR |
Abbreviations: NR, not reported; C, centrifugation; UC, ultracentrifugation; CLSM, confocal laser scanning microscopy; ESCRT, Endosomal Sorting Complex Required for Transport; FC, flux cytometry; DLS, dynamic light scattering; NTA, Nanoparticle tracking analysis; PA, proteomic analysis; SEM, scanning electronic microscopy; SG, sucrose gradient; TEM transmission electronic microscopy.
eYeast extract, glucose, ascorbic acid, cysteine, ferric ammonium citrate, vitamins, salts and bovine serum pH 6.8.
fTrypticase, yeast‐extract, glucose, ascorbic acid, cysteine, ferric ammonium citrate, vitamins, salts and bovine serum, pH 6.8.