EV-TRACK

Search > Results

You searched for: EV190050 (EV-TRACK ID)

Showing 1 - 1 of 1

Results list Study Tree

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190050	1/1	Mus musculus	4T1	DG Filtration SEC SEC (non-commercial)	Ger J A Arkesteijn	2020	63%
Study summary Full title Improved Flow Cytometric Light Scatter Detection of Submicron-Sized Particles by Reduction of Optical Background Signals All authors Ger J A Arkesteijn, Estefanía Lozano-Andrés, Sten F W M Libregts, Marca H M Wauben Journal Cytometry A Abstract Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of (show more...) Flow cytometry allows multiparameter analysis on a single-cell basis and is currently the method of choice to rapidly assess heterogeneity of cell populations in suspension. With the research field of extracellular vesicles (EV) rapidly expanding, there is an increased demand to address heterogeneity of EV populations in biological samples. Although flow cytometry would be the ideal technique to do so, the available instruments are in general not equipped to optimally detect the dim light scatter signals generated by submicron-sized particles like EV. Although sideward scatter light and fluorescence are currently used as a threshold signal to identify EV within samples, the forward scatter light (FSC) parameter is often neglected due to the lack of resolution to distinguish EV-related signals from noise. However, after optimization of FSC detection by adjusting the size of the obscuration bar, we recently showed that certain EV-subsets could only be identified based on FSC. This observation made us to further study the possibilities to enhance FSC-detection of submicron-sized particles. By testing differently sized obscuration bars and differently sized pinholes in the focal plane behind the FSC detection lens, we generated a matrix that allowed us to determine which combination resulted in the lowest optical background in terms of numbers of events regarding FSC detection of submicron-sized particles. We found that a combination of an 8-mm obscuration bar and a 200-μm pinhole reduced optical background in a reproducible manner to such extent that it allowed a robust separation of 100-nm polystyrene beads from background signals within the FSC channel, and even allowed thresholding on FSC without the interference of massive background signals when both beads and EV were measured. These technical adaptations thus significantly improved FSC detection of submicron-sized particles and provide an important lead for the further development and design of flow cytometers that aid in detection of submicron-sized particles. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry. (hide) EV-METRIC 63% (93rd 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 breast tumor model Focus vesicles extracellular vesicle 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 DG Filtration SEC Size-exclusion chromatography (non-commercial) Protein markers EV: TSG101/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ Flotillin2/ HSP70/ MHC2/ CD9/ MHC1 non-EV: Proteomics no EV density (g/ml) 1.10-1.12 Show all info Study aim Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells 4T1 EV-harvesting Medium EV-depleted medium Preparation of EDS >=18h at >= 100,000g Cell viability (%) 90 Separation Method Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 5% Highest density fraction 40% Total gradient volume, incl. sample (mL) 16.5 Sample volume (mL) 1 Orientation Top-down Rotor type SW 32.1 Ti Speed (g) 100000 Duration (min) 1091 Fraction volume (mL) 1 Fraction processing Size-exclusion chromatography Filtration steps 0.45µm > x > 0.22µm, Size-exclusion chromatography Total column volume (mL) 10 Sample volume/column (mL) 2 Resin type Sepharose CL-2B Other Name other separation method Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins CD9/ CD63 Not detected EV-associated proteins HSP90/ HSP70/ MHC1/ CD81/ Flotillin1/ TSG101/ MHC2/ Flotillin2/ Alix Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Mean Reported size (nm) 139.8 EV concentration Yes

				1 - 1 of 1

EV-TRACK ID	EV190050
species	Mus musculus
sample type	Cell culture
cell type	4T1
condition	breast tumor model
separation protocol	DG Filtration SEC Size-exclusion chromatography (non-commercial)
Exp. nr.	1
EV-METRIC %	63