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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV210158	1/2	Bos taurus	Mac-T cells	(d)(U)C Filtration	Ogunnaike, Mojisola	2021	67%
Study summary Full title Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery All authors Mojisola Ogunnaike, Haichuan Wang, Janos Zempleni Journal Int J Pharma Abstract Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BM (show more...)Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies. (hide) EV-METRIC 67% (94th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 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. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: Calnexin/ Histone H3 Proteomics no Show all info Study aim Exosomes and cargo characterization Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells Mac-T cells EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 97 Cell count 6,20E+06 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8100 rotor Pelleting: speed (g) 130000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants Histone H3/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 100 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 94 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 6,11E+08 EM EM-type Transmission-EM Image type Wide-field Report size (nm) ~90

	EV210158	2/2	Bos taurus	Bovine milk	(d)(U)C Filtration	Ogunnaike, Mojisola	2021	67%
Study summary Full title Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery All authors Mojisola Ogunnaike, Haichuan Wang, Janos Zempleni Journal Int J Pharma Abstract Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BM (show more...)Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies. (hide) EV-METRIC 67% (87th 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. For the quantitative method, the reporting of measured EV concentration is expected. 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 Bovine milk 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. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (d)(U)C Filtration Protein markers EV: TSG101/ CD81/ CD63/ CD9 non-EV: Calnexin/ Histone H3 Proteomics no Show all info Study aim Exosomes and cargo characterization Sample Species Bos taurus Sample Type Bovine milk Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: time(min) 90 Pelleting: rotor type F37L-8100 rotor Pelleting: speed (g) 130000 Filtration steps 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63/ TSG101/ CD81 Not detected contaminants Histone H3/ Calnexin Characterization: RNA analysis RNA analysis Type (RT)(q)PCR Database No Proteinase treatment Yes Moment of Proteinase treatment After Proteinase type Proteinase K Proteinase concentration 100 RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 101 EV concentration Yes Particle yield Yes, as number of particles per milliliter of starting sample 3,40E+10 EM EM-type Transmission-EM Image type Wide-field Report size (nm) ~100

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EV-TRACK ID	EV210158
species	Bos taurus
sample type	Cell culture	Bovine milk
cell type	Mac-T cells	NA
medium	EV-depleted medium	NA
condition	Control condition	Control condition
separation protocol	(d)(U)C Filtration	(d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	67	67