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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220127	1/2	Homo sapiens	lung tissue	(d)(U)C DG Filtration	Liu, Bowen/ Jin, Yuan	2022	100%
Study summary Full title Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation All authors Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide) EV-METRIC 100% (80th 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 lung tissue Sample origin Control condition 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 20553 (pelleting) / 17842 (washing) Protein markers EV: Alix/ CD9/ CD81 non-EV: Albumin/ Argonaute-2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.1 Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type lung tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 20553 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 110,000 Wash: adjusted k-factor 17842 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 0.25 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.45 Orientation Bottom-up Rotor type MLS-50 Speed (g) 180,000 Duration (min) 780 Fraction volume (mL) 0.45 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 17842 Pelleting-wash: volume per pellet (mL) 1.5 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) TLA-55 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.8 Western Blot Detected EV-associated proteins Alix/ CD9/ CD81 Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 131.7 Particle analysis: flow cytometry Flow cytometer type BD LSRFortessa Hardware adjustment use calibration beads Calibration bead size 0.05/ 0.1/ 0.2/ 0.3/ 0.5 Report type Size range/distribution Reported size (nm) 100 - 200 EM EM-type Cryo-EM Image type Close-up, Wide-field

	EV220127	2/2	Mus musculus	lung tissue	(d)(U)C DG Filtration	Liu, Bowen/ Jin, Yuan	2022	100%
Study summary Full title Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation All authors Bowen Liu, Yuan Jin, Jingyi Yang, Yue Han, Hui Shan, Mantang Qiu, Xuyang Zhao, Anhang Liu, Yan Jin, Yuxin Yin Journal J Extracell Vesicles Abstract Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range (show more...)Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications. (hide) EV-METRIC 100% (80th 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 lung tissue Sample origin Control condition 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 (Differential) (ultra)centrifugation Density gradient Filtration Adj. k-factor 20553 (pelleting) / 17842 (washing) Protein markers EV: Alix/ CD9/ Flotillin-1/ TSG101 non-EV: GM130/ Calnexin/ Albumin/ Argonaute-2/ Calreticulin/ PMP70/ Prohibitin/ Tamm-Horsfall protein Proteomics yes EV density (g/ml) 1.1 Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Mus musculus Sample Type lung tissue Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type Type 45 Ti Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 20553 Wash: volume per pellet (ml) 1.5 Wash: time (min) 70 Wash: Rotor Type TLA-55 Wash: speed (g) 110,000 Wash: adjusted k-factor 17842 Density gradient Type Discontinuous Number of initial discontinuous layers 10 Lowest density fraction 0.25 M Highest density fraction 2.5 M Total gradient volume, incl. sample (mL) 4.5 Sample volume (mL) 0.45 Orientation Bottom-up Rotor type MLS-50 Speed (g) 180,000 Duration (min) 780 Fraction volume (mL) 0.45 Fraction processing Centrifugation Pelleting: volume per fraction 1.5 Pelleting: speed (g) 110,000 Pelleting: adjusted k-factor 17842 Pelleting-wash: volume per pellet (mL) 1.5 Pelleting-wash: duration (min) 70 Pelleting-wash: speed (g) TLA-55 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Protein Yield (µg) 0.7 Western Blot Detected EV-associated proteins Alix/ CD9/ Flotillin-1/ TSG101 Not detected contaminants GM130/ Calnexin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 124.9 Particle analysis: flow cytometry Flow cytometer type BD LSRFortessa Hardware adjustment use calibration beads Calibration bead size 0.05/ 0.1/ 0.2/ 0.3/ 0.5 Report type Size range/distribution Reported size (nm) 100 - 200 EM EM-type Cryo-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV220127
species	Homo sapiens	Mus musculus
sample type	lung tissue	lung tissue
condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
Exp. nr.	1	2
EV-METRIC %	100	100