TABLE 3.

Studies on Toxoplasma gondii 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
Aline et al., 2004	Exosomes excreted by DCs infected with tachyzoites	1 × 106 DC cells/mL	DCs cultured for 18 h and supernatant recovered	NR	C ‐ 3000 × g/30 min 10,000 × g/30 min	UC‐ 100,000 × g/60 min Pellets resuspended in PBS (200 μL) containing protease inhibitors.	TEM Protein concentration	NR	Immunization	NR
Beauvillain et al., 2007	Exosomes excreted by DCs treated with tachyzoites antigen	NR	DC supernatant recovered	NR	C ‐ 3000 × g/30 min 10,000 × g/30 min	UC −100,000 × g/60 min	TEM Protein concentration	Exosomes‐10 μg	Immunization	NR
Beauvillain et al., 2009	Exosomes excreted by DCs infected with tachyzoites	NR	DC supernatant recovered	NR	C ‐ 3000 × g/30 min 10,000 × g/30 min	UC 100,000 × g/ 60 min	TEM Protein concentration	NR	Immunization	NR
Pope & Lasser, 2013	HFF infected with tachyzoites in DMEM 10% FBS	NR	Tachyzoites in FBS free DMEM used to induce exosome in incubation period of 24 and 72‐h	NR	C (4°C). 300 × g/10 min 16,500 × g/20 min	UC 140,000 × g/45 min. EVs resuspended in 200 μL PBS	TEM	NR	Microarray miRNA isolation	NR
Kim et al., 2016	L6 cells infected with tachyzoites	5 × 105 L6 cells infected with tachyzoites	Infected L6 cells supernatant recovered after 12 h	NR	C 300 × g/10 min 2000 × g/10 min 10,000 × g/30 min	UC in 2 steps of 100,000 × g/70 min. Pellet resuspended in 300 μL of PBS.	FC	NR	miRNA microarray	NR
Wowk et al., 2017	HFF cells in DMEM infected with tachyzoites	HFF cells infected with tachyzoites (MOI 10)	Incubation of parasites in FBS free DMEM 2 h/37°C EV‐containing supernatants filtrated in 0.45 μm sterile cartridges	Observation of tachyzoites viability and concentration	C to remove parasites	Total Exosome Isolation kit (Invitrogen)	NTA TEM Protein concentrations in Quibit fluorometric quantification	NR	PA	NR
Silva et al., 2018	VERO cells in DMEM 10% FBS infected with tachyzoites	NR	Tachyzoites in FBS free DMEM (1 mL) incubated at 37°C in different times (1, 3, 6, 12, 20 and 24 h). EV‐containing supernatants filtrated in 0.22‐μm sterile cartridges	In TEM	C – 3500 × g/10 min.	SEC	NTA TEM SEM Protein concentration	NR	EV proteins analysis and immunoblotting miRNA isolation Cytokine assay	NR
Li et al., 2018b	HFF infected with tachyzoites	100 mL of supernatant containing tachyzoites	Tachyzoites maintaining in FBS free DMEM (100 mL) for 24 h EV‐containing supernatants filtered in 0.22 μm sterile cartridges and concentrated in a filter unit of Vivaflow 100 kDa MWCO PES (EMD Millipore, Billerica, MA, USA)	TEM	C ‐ 5000 × g/10 min	ExoEasy Maxi Kit (Qiagen, Hilden, Germany).	NTA TEM FC	80 μg of EVs from × in 8 106 tachyzoites DMEM (100 mL)	Macrophage cytokine assay Immunization and challenge Uptake of T. gondii exosomes by macrophages	CD63 P30 HSP70
Li et al., 2018a	DC2.4 in 10% EVdepleted FBS RPMI infected with tachyzoites	DC2.4 cells infected with tachyzoites (MOI 3) for 28 h	Supernatants collected for exosome isolation. EV‐containing supernatants filtrated with a 0.22 μm sterile cartridges	In TEM	C ‐ 2000 × g/20 min 10,000 × g/30 min	UC (4°C) 100,000 × g/60 min Pellet resuspended in PBS (0.1 mL)	NTA TEM Exosomal proteins Immunoblotting	NR	Total and small RNA extraction RNA sequencing miRNA sequencing Analysis of the differential exosomal miRNAs	CD63 TSG101
Ramirez‐Flores et al., 2019	MDCK cells cultured in coverslips with DMEM and infected with tachyzoites	3 × 108 tachyzoites/mL	Tachyzoites in sterile PBS incubated at 4 h (37°C) EV‐containing supernatants filtrated with a 0.22 μm sterile cartridges	Tachyzoites viability determined by the exclusion of SYTOX‐acid green nucleic stain	C at 4°C 200 × g/10 min 10,000 × g/10 min	UC 150,000 × g at 4°C twice	NTA TEM Protein concentration Immunoblotting	NR	Molecular and morphological identification TEM SEM Ultrastructure EVs: Immune‐recognition Self‐assembled tubular structures	Lamin B1 α‐Tubulin TgARO H3 β‐Tubulin
da Cruz et al., 2020	Human EVs purified of CSF and sera from patients with toxoplasmosis	CSF: 600 μL Serum:300 μL	NR	TEM	C 13,500 × g/15 min	UC 100,000 × g/60 min at 25°C. Pellets, containing EVs, resuspended in 100 μL of filtered PBS.	NTA TEM Protein concentration Immunoblotting	Toxoplasmosis patients: mean of 2.4 ×1010 EVs/mL Seronegative individuals: mean of 5.9 × 109 EVs/mL	Human miRNA expression	CD63 CD9
Maia et al., 2021a	VERO cells in DMEM 10% FBS infected with tachyzoites	NR	Tachyzoites in FBS free RPMI 1640 (1 mL) incubated for 2 h/37°C. EV containing supernatants filtrated with a 0.22 μm sterile cartridges.	TEM	C (5x) 2500 × g/10 min After vesiculation: Centrifugation 3500 × g/10 min Murine sera centrifugation 13,500 × g/15 min	SEC Murine sera (1 mL) Ultracentrifugation 100,000 × g/60 min (25°C)	NTA TEM Protein concentration Immunoblotting	Mice: pooled serumderived EVs (1 mL): Normal: 2.22 × 1010 EVs/mL Immunized: 8.40 × 1010 EVs/mL) Infected: 8.92 × 1010 EVs/mL	Murine miRNA expression Murine cytokine determination	CD63 CD9
Maia et al., 2021b	VERO cells in DMEM 10% FBS infected with tachyzoites	1 × 109 tachyzoites	Tachyzoites in FBS free RPMI 1640 (1 mL) incubated for 2 h/37°C. EV containing supernatants filtrated with a 0.22 μm sterile cartridges	TEM	C (5x) 2500 × g/10 min After vesiculation: Centrifugation 3500 × g/10 min	SEC	NTA BCA TEM Protein concentration Immunoblotting	2.55 × 109 EVs/mL	Immunizations and challenge Stimulation of cellular and humoral responses	NR
Quiarim et al., 2021	VERO cells in DMEM 10% FBS infected with 3 T. gondii strains tachyzoites	NR	Tachyzoites, of each T. gondii strain, in FBS free DMEM (1 mL) incubated for 2 h/37°C. EV containing supernatants filtrated with a 0.22 μm sterile cartridges	TEM	‐T. gondii EVs from culture supernatants were washed 5 times (2500 × g/5 min) and filtered.	SEC	NTA TEM Immunoblotting SEM	EV released in 2 h for each strain: 8.0 × 108 (RH), 1.9 × 108.(ME49), 4.8 × 108 (VEG) EV released in 24 h for the 3 strain: around 1.2 × 108	miRNA isolation by reverse transcription and quantitative realtime. Murine cytokine determination Virulence in mice	NR

Abbreviations: NR, not reported; C, centrifugation; UC, ultracentrifugation; FC, flow cytometry; NTA, Nanoparticle tracking analysis; PA, proteomic analysis; SEM, scanning electronic microscopy; TEM, transmission electronic microscopy.