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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220366	1/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (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 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 Protein markers EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 5.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	3/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (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 Ionomycin 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 Protein markers EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 7.50E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	4/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	67%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (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 compound 634 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 Protein markers EV: Alix/ CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: Calnexin Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins Alix/ CD81/ CD86/ CD80/ MHCII Not detected EV-associated proteins CD40 Detected contaminants Calnexin Characterization: Lipid analysis No Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 75-300 EV concentration Yes Particle yield particles per milliliter of starting sample: 8.00E+11 EM EM-type Transmission-EM Image type Wide-field

	EV220366	2/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	44%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 44% (84th 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 MPLA 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 Protein markers EV: CD81/ CD86/ CD80/ MHCII/ CD40 non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) per milliliter of starting sample Western Blot Detected EV-associated proteins CD81/ CD86/ CD80/ MHCII/ CD40 Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	5/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2E241 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	6/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2F186 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	7/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2H013 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	8/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2G176 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	9/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2G179a 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	10/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2G179c 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Protein Yield (µg) per milliliter of starting sample Characterization: Lipid analysis No Characterization: Particle analysis None

	EV220366	11/11	Mus musculus	bone marrow-derived cells	(d)(U)C	Sako Y	2023	14%
Study summary Full title Identification of a Novel Small Molecule That Enhances the Release of Extracellular Vesicles with Immunostimulatory Potency via Induction of Calcium Influx. All authors Sako Y, Sato-Kaneko F, Shukla NM, Yao S, Belsuzarri MM, Chan M, Saito T, Lao FS, Kong H, Puffer M, Messer K, Pu M, Cottam HB, Carson DA, Hayashi T Journal ACS Chem Biol Abstract Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapse (show more...)Extracellular vesicles (EVs) transfer antigens and immunomodulatory molecules in immunologic synapses as a part of intracellular communication, and EVs equipped with immunostimulatory functions have been utilized for vaccine formulation. Hence, we sought small-molecule compounds that increase immunostimulatory EVs released by antigen-presenting dendritic cells (DCs) for enhancement of vaccine immunogenicity. We previously performed high-throughput screening on a 28K compound library using three THP-1 reporter cell lines with CD63 Turbo-Luciferase, NF-κB, and interferon-sensitive response element (ISRE) reporter constructs, respectively. Because intracellular Ca elevation enhances EV release, we screened 80 hit compounds and identified compound as a Ca influx inducer. enhanced EV release in murine bone marrow-derived dendritic cells (mBMDCs) and increased costimulatory molecule expression on the surface of EVs and the parent cells. EVs isolated from -treated mBMDCs induced T cell proliferation in the presence of antigenic peptides. To assess the roles of intracellular Ca elevation in immunostimulatory EV release, we performed structure-activity relationship (SAR) studies of . The analogues that retained the ability to induce Ca influx induced more EVs with immunostimulatory properties from mBMDCs than did those that lacked the ability to induce Ca influx. The levels of Ca induction of synthesized analogues correlated with the numbers of EVs released and costimulatory molecule expression on the parent cells. Collectively, our study presents that a small molecule, , enhances the release of EVs with immunostimulatory potency via induction of Ca influx. This agent is a novel tool for EV-based immune studies and vaccine development. (hide) EV-METRIC 14% (43rd 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 compound 2G179g 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 Protein markers EV: None non-EV: None Proteomics no Show all info Study aim Function Sample Species Mus musculus Sample Type Cell culture supernatant EV-producing cells bone marrow-derived cells EV-harvesting Medium EV-depleted medium Preparation of EDS Commercial EDS Cell count 3.00E+07 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: rotor type SW 28 Pelleting: speed (g) 100,000 Wash: volume per pellet (ml) 30 Wash: time (min) 180 Wash: Rotor Type SW 28 Wash: speed (g) 100,000 Characterization: Protein analysis None Protein Concentration Method Not determined Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV220366
species	Mus musculus
sample type	Cell culture
cell type	bone marrow-derived cells
condition	Control condition	Ionomycin	compound 634	MPLA	compound 2E241	compound 2F186	compound 2H013	compound 2G176	compound 2G179a	compound 2G179c	compound 2G179g
separation protocol	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC	dUC
Exp. nr.	1	3	4	2	5	6	7	8	9	10	11
EV-METRIC %	67	67	67	44	14	14	14	14	14	14	14

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