TABLE 5.

Studies on Protista EVs.

Reference	Vesicle cellular origin	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
Sharma et al., 2020	Entamoeba histolytica trophozoites	NR	16 h‐incubation at 35.5°C/anaerobiosis in serum‐free TYI‐S‐33 a (10 mL)	NR	C (2000 rpm) and filtration (0.22 mm)	Total Exosome Isolation reagent	TEM, NTA, Small RNA cargo analysis	125 nm (NTA) Concentration NR	PA EVs signalizing in encystation	Lgl
Díaz‐Godínez et al., 2022	Entamoeba histolytica trophozoites	2.5 × 105/mL	1 h‐incubation at 37°C in RPMI (3 mL)	NR	C (1400 rpm, 5 min) and filtration (0.22 mm)	Total Exosome Isolation reagent	TEM, NTA, Lipid staining (DiO dye)	167 nm (NTA) Concentration NR	PA Neutrophil uptake Neutrophil NETs and ROS assays	NR
Galindo et al., 2022	Entamoeba histolytica trophozoites	NR	NR	NR	C (10,000 rpm, 30 min)	UC (100,000 × g 70 min)	TEM	68‐110 nm (TEM) Concentration NR	Labelling with antiVps23 antibody	EhVps23
Costa et al., 2021	Acanthamoeba trophozoites	5 × 106/mL	2 h‐incubation at 37oC, 180 rpm in serum free RPMI (8 mL)	>93%	C (700 × g, 10 min; 2000 × g, 20 min) and filtration (0.22 mm)	UC (100,000 × g 1 h)	TEM, SEM, NTA	101‐150 nm (NTA) 1.2 x 109 to 8.6 × 1011/mL	Zymography Murine macrophages stimulation (cytokines production)	NR
Lin et al., 2020	Acanthamoeba trophozoites	NR	48 h‐ incubation at 28°C in PAS b (volume NR)	NR	C (2000 × g 30 min; 10,000 × g, 30 min) and filtration (0.22 mm)	Total Exosome Isolation reagent	TEM, NTA	118 nm (NTA) 2.83 × 1010/mL	PA Rat glial C6 cells uptake and cytotoxicity THP‐1 cells stimulation (cytokines transcription) Aminopeptidase activity	NR
Retana‐Moreira et al., 2019	Acanthamoeba trophozoites	4 × 106 in the total	24 h‐incubation at 28°C and 37°C in PYG c (volume NR)	>99%	C (3500 × g, 15 min; 17,000 × g, 15 min) and filtration (0.22 mm)	UC (100,000 × g 18 h)	DLS, AFM	184 nm (28°C) 111 nm (37°C) Concentration NR	Zymography	NR
Gonçalves et al., 2018	Acanthamoeba trophozoites	NR	48 h‐incubation at 28°C in PYG c and glucose medium d (volume NR)	NR	Concentration in 10 KDa cut‐off membrane, C (1100 × g, 10 min) and filtration (0.8 mm)	UC (100,000 × g 1 h)	TEM, SEM, DLS	117 nm (TEM) 150‐235 nm (DLS) Concentration NR	PA Lipidomic (GC‐MS) Zymography CHO and T98G cells uptake and cytotoxicity	NR
Lertjuthaporn et al., 2022	Naegleria fowleri trophozoites	NR	24 h‐incubation at 37°C in PAS b (volume NR)	75 ± 9.41%	C (1500 × g, 15 min) and filtration (1.2 mm)	C (21,000 × g, 70 min), fil‐tration (0.22 mm) and UC (110,000 × g 1.5 h)	TEM, NTA	∼158 nm‐ NfMP ∼141 nm—NfExo (NTA) ∼4.6 x 108/mL	Uptake by THP‐1 macrophages THP‐1 macrophages stimulation (costimulatory molecules expression and inflammatory cytokines production)	NR
Retana Moreira et al., 2022	Naegleria fowleri trophozoites	5 × 106/mL	5 h‐incubation at 37°C in serum‐free 2% casein hydrolysate medium (10 mL)	97.5%	C (3500 × g, 15 min; 17,000 × g, 15 min) and filtration (0.22 mm)	UC (120,000 × g 2.5 h)	Protein Micro BCA assay, NTA, TEM, AFM, DLS	206 nm (NTA) 4.96 × 1010/mL	PA Zymography Immunoproteomic (using polyclonal antiN. fowleri antibodies)	NR

Abbreviations: NR, not reported; C, centrifugation; UC, ultracentrifugation; AFM, atomic force microscopy; NTA, Nanoparticle tracking analysis; DLS, dynamic light scattering; PA, proteomic analysis; SEM, scanning electronic microscopy; TEM, transmission electronic microscopy.

^a Trypticase, yeast‐extract, glucose, ascorbic acid, cysteine, ferric ammonium citrate, vitamins, salts and bovine serum, pH 6.8.

^b Pageʼs modified Neffʼs amoeba saline (only salts).

^c Proteose‐peptone, yeast extract, glucose, and salts, pH 6.5.

^d Glucose and salts (Proteose‐peptone‐free and yeast extract‐free PYG).