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Results list Most used separation protocols Top EV-enriched proteins

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	separation protocol	First author	Year	EV-METRIC
	EV190103	1/1	Homo sapiens	Cell culture supernatant	DG (d)(U)C Dynabeads CD9, CD63, CD81	Xiaoju Zhou	2020	100%
Study summary Full title Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing All authors Xiaoju Zhou, Brooke A Brown, Amanda P Siegel, Mohamed El Masry, Xuyao Zeng, Woran Song, Amitava Das, Puneet Khandelwal, Andrew Clark, Kanhaiya Singh, Poornachander R Guda, Mahadeo Gorain, Lava Timsina, Yi Xuan, Stephen C Jacobson, Milos V Novotny, Sashwati Roy, Mangilal Agarwal, Robert J Lee, Chandan K Sen, David E Clemmer, Subhadip Ghatak Journal ACS Nano Abstract Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a (show more...)Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA, has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP reporter (Exoκ-GFP) using tissue nanotransfection and were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low base pair RNA and were selectively engulfed by wound-macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFP in vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, TLNPκ/si-hnRNPA2B1 treated group showed significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP are recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties post-injury. (hide) EV-METRIC 100% (99th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Cell Name HaCaT Sample origin Control condition Focus vesicles exosome Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. (d)(U)C = (differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C Dynabeads CD9, CD63, CD81 Protein markers EV: TSG101/ Alix non-EV: Proteomics no EV density (g/ml) 1.16 Show all info Study aim Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer Sample Species Homo sapiens Sample Type Cell culture supernatant Sample Condition Control condition EV-producing cells HaCaT EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell viability Yes Cell viability (%) Yes Separation Method Differential ultracentrifugation centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Obtain an EV pellet : Yes Pelleting: time(min) 120 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Wash: volume per pellet (ml) 0.5 Wash: time (min) 120 Wash: Rotor Type TLA-120.2 Wash: speed (g) 245000 Density gradient Only used for validation of main results Yes Density medium Sucrose Type Discontinuous Number of initial discontinuous layers 6 Lowest density fraction 0.4M Highest density fraction 2.5M Total gradient volume, incl. sample (mL) 1.05 Sample volume (mL) 0.05 Orientation Top-down Rotor type TLA-120.2 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 1 Pelleting: duration (min) 120 Pelleting: rotor type TLA-120.2 Pelleting: speed (g) 245000 Commercial kit Dynabeads CD9, CD63, CD81 Characterization: Protein analysis Protein Concentration Method microBCA Western Blot Detected EV-associated proteins TSG101/ Alix/ FLOT1/ HSP90 Detected contaminants Prohibitin Not detected contaminants GM130 Flow cytometry specific beads Detected EV-associated proteins TSG101 Other 1 Fluorescent anisotropy Detected EV-associated proteins TSG101 Characterization: Particle analysis NTA Report type Modus Reported size (nm) 102 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3.20E+07 EM EM-type Scanning-EM Image type Wide-field

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