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Ribovski et al. Extracell Vesicles Circ Nucleic Acids 2023;4:283-305 https://dx.doi.org/10.20517/evcna.2023.26 Page 9

Table 1. Assays to study uptake mechanisms, intracellular localization, and functional cargo delivery by EVs

EVs Producer cells or source material Recipient cells Assay type Uptake mechanism Reference
[24]
EV-GLA Stable clones of CHO DG44 expressing GLA and Primary cultures of mouse aortic endothelial Pharmacological inhibitors of Clathrin-mediated endocytosis
HEK293T cells (MAEC) derived from Fabry KO mice and endocytosis; and macropinocytosis
HEK293 cells DiOC, DiD, or DiR labeled EVs
[25]
placenta-derived sEVs Primary human trophoblasts, Primary placental fibroblasts (PPF) and Pharmacological inhibitors of Clathrin-mediated endocytosis
Stable clones BeWo cells transfected with mCherry- primary human uterine microvascular endocytosis; and macropinocytosis
TSG101-NanoLuc endothelial cells (HUtMEC) siRNA-mediated knockdown of
specific pathways;
NanoLuc activity;
DiI-labeled EVs loaded with
NanoLuc protein;
Alexa488-labelled microRNA-
517a
[70]
DSP1-Tetraspanin EVs Stable clones of SUM159 cells transfected with DSP2 DSP2-expressing SUM159 cells and SUM159 Pharmacological inhibitors of Dynamin2-
and DSP1-CD9 cells endocytosis; dependent/Clathrin-mediated
DSP reporter proteins (EGFP and endocytosis
luciferase);
Colocalization with EEA1 and
CD63
[113]
grapefruit-derived EVs Grapefruit juice HaCaT cells Pharmacological inhibitors of Clathrin-mediated endocytosis
endocytosis;
DiO-labeled EVs;
TAMRA-labeled siRNA;
siRNA-mediated gene
knockdown
[59]
NLuc-Hsp70-EVs, Stable clone of HeLa cells transfected with NLuc-Hsp70, HeLa cells, HEK293T cells, HEK293T Colocalization with Rab5 and Membrane fusion
NLuc-CD63-EVs, Nluc-HsP70 or GFP-Hsp70 and of HEK293T transfected overexpressing Flag-tagged IFITM1 or 3 Lamp1;
GFP-Hsp70 EVs with flag-tagged IFITM1 and IFITM3 Cytosolic and membrane
fraction separation + NLuc
activity;
Pharmacological inhibitor
(BafA1);
Inhibition by IFITM proteins
(fusion)
[72]
Syn1-EVs,VSV-G EVs Stable clone HeLa cells transfected with NLuc-Hsp70 or HeLa cells, GFP-PEST HT1080 acceptor cells NLuc activity; Membrane fusion
carrying FRB-NLuc-HA or CD8-GFP, Syncytia formation
NLuc-Hsp70 HT1080 and HeLa cells overexpressing Syn1 and VSV-G
[64]
Normoxic U87 and GL261 Normoxic U87 MG or GL261 cells U87 MG and GL261 cells, and primary patient- Colocalization with transferrin, Raft–mediated endocytosis
EVs derived human GBM cell lines U3034, U3065, dextran, cholera toxin B (raft (heparan sulfate proteoglycan
and U3082 (HGG cells) marker); endocytosis)
PKH67 green or PKH26 red-
labeled EVs

GLA: alpha-galactosidase A; Syn1: Syncytin-1; BafA1: bafilomycin A1; Nluc: NanoLuc luciferase; DSP: Dual Split; NanoLuc luciferase-tagged Hsp70: NLuc-Hsp70; FRB: FKBP-rapamycin binding; HGG: high-grade