Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Specific recognition of hyaluronan by the Ig-like-V-set domain of hSiglec-9. (a) Immobilized high molecular weight-hyaluronan (HMW-HA) was probed with human Siglec–Fc and CD44–Fc chimeras and binding evaluated by using an anti-human IgG-HRP. (b) Binding of hSiglec-9–Fc to immobilized HMW-HA was performed in the presence of increasing concentrations of HMW-HA, heparan sulfate, chondroitin sulfate or heparin. (c, d) To map the hSiglec-9 domain responsible for HA recognition, binding of hSiglec-9–Fc to immobilized HMW-HA was compared to binding of hSiglec-9–Fc with an Arg→Ala mutation in the V-set domain (hSiglec-9R120K), a fusion protein construct of the hSiglec-9 V-set domain + the second Ig-like domain (C2-set) of hSiglec-7 + human IgG Fc tail (hSiglec-9V-7C2–Fc), a fusion protein construct encompassing V-set domain of hSiglec-7 + C2-set of hSiglec-9 + human IgG Fc tail (Siglec-7V-9C2–Fc), a fusion protein construct of the hSiglec-9 V-set domain + first C2-set domain + human IgG Fc tail (Siglec-9–Fc 2D). Results are expressed as the mean ± SD. All experiments were performed in triplicate, repeated 3 times (a, d) or 2 times (b). One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Mutagenesis, Construct, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: High molecular weight hyaluronan (HMW-HA) is recognized by hSiglec-9 on human neutrophils. (a) To evaluate the molecular size of hyaluronan (HA) responsible for binding to hSiglec-9, high molecular weigh HA (HMW-HA; >1,000 kDa), low molecular weigh (200 – 300 kDa-HA and 25 – 75 kDa-HA), nano-HA (nonamers) and oligo-HA (hexamers) fragments were added to compete with binding of hSiglec-9–Fc to immobilized HMW-HA plates. Binding was evaluated using an anti-human IgG-HRP. Experiment performed in triplicate and repeated 3 times; results are expressed as mean ± SD. (b) Flow cytometry reveals constitutive expression of Siglec-9 and CD44 on human neutrophils from 9 different donors; geometric mean ± 95% confidence interval. (c) Binding of FITC-labeled HMW-HA to neutrophils of these donors was evaluated by flow cytometry; geometric mean ± 95% confidence interval. (d) Human neutrophils were pretreated with anti-Siglec-9 monoclonal antibodies, anti-human CDw329 (BD Pharmingen, #550906) and anti-human Siglec-9 (R&D Systems, #BAF1139) and effects on binding to FITC-labeled HMW-HA determined. CDw329 Ab blocked binding of HMW-HA, but not GBS capsule (Fig. 5b) and was designated “α-Sig-9(HA)”; in contrast, the R&D Systems Ab blocked recognition of GBS capsule (Fig. 5b), but did not interfere with binding to HMW-HA, and was thus designated “α-Sig-9(Sia)”. Data pooled from five independent experiments in triplicate; data represent the mean ± SD. (e, f) Calcein-labeled human neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) and α-CD44 mAbs, added to wells coated with immobilized HMW-HA to facilitate adherence, then unbound neutrophils washed away. Remaining neutrophils were lifted and enumerated. Experiment performed in triplicate and repeated 5 times; results are expressed as mean ± SD. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Molecular Weight, Binding Assay, Flow Cytometry, Expressing, Labeling, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) engages hSiglec-9 via its surface hyaluronan capsule. (a) Human Siglec-9–Fc was immobilized to ELISA wells using protein A, and binding of FITC-labeled forms of WT GAS, its isogenic HA capsule-deficient mutant (ΔhasA), an animal-passaged hyperencapsulated (AP) derivative and its isogenic HA capsule-deficient mutant (AP ΔhasA) were evaluated; results are expressed as mean ± SD and repeated 5 times in triplicate with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) (b) Human Siglec-9–Fc chimera was immobilized to ELISA wells via protein A in the presence of α-Sig-9(HA), α-Sig-9(Sia) or isotype control Abs and binding of FITC-labeled WT GAS or sialic acid-expressing serotype III group B Streptococcus (GBS) evaluated. (c) Human Siglec-9/9R120K/5/6/7/11 and human CD44-Fc chimera were immobilized to ELISA wells via protein A. Binding of FITC-labeled WT GAS was evaluated. Results represent mean ± SD; triplicate wells, representative experiment depicted of 5 independent repeats with similar results, performed in triplicate. (d) hSiglec-9–Fc was immobilized to ELISA plates using protein A, then wells were pretreated with HMW-HA, HMW-heparan sulfate or heparin over the indicated range of concentrations. Binding of FITC-labeled WT GAS was evaluated.

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Labeling, Mutagenesis, Comparison, Expressing

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: HMW-HA binding to hSiglec-9 induces SHP-1 recruitment and blunts neutrophil NET production and oxidative burst. (a) 1.2 × 107 human neutrophils were incubated for 30 min + 10 µg/ml of high molecular weight hyaluronan (HMW-HA) + 25 nM PMA. Cell lysates were immunoprecipitated with α-Siglec9 and SHP-1 recruitment was visualized by western blot analysis; results were repeated 2 times with similar results; representative experiment with relative densitometry values is shown. (b) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 Abs, incubated with 10 µg/ml of HMW-HA and activated for 30 min with PMA. Reactive oxygen species (ROS) release was measured with the OxyBURST Green H2HFF BSA probe and results expressed as mean fluorescence intensity (MFI) ± SD; experiment repeated 5 times with similar results; representative experiment is shown. (c) Neutrophils were pretreated α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs, then incubated with 10 µg/ml of HMW-HA and activated with PMA for 3 h; production of neutrophil extracellular traps (NETs) visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); representative fields at 20x magnification is shown; experiment performed in triplicate and repeated 5 times. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD; experiment repeated 3 times in triplicate with similar results; representative experiment shown. One-way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Incubation, Molecular Weight, Immunoprecipitation, Western Blot, Fluorescence, Staining, Picogreen Assay, Comparison

Journal: Journal of molecular medicine (Berlin, Germany)

Article Title: Host and Pathogen Hyaluronan Signal Through Human Siglec-9 to Suppress Neutrophil Activation

doi: 10.1007/s00109-015-1341-8

Figure Lengend Snippet: Group A Streptococcus (GAS) binding to hSiglec-9 via its surface HMW-HA capsule blunts neutrophil oxidative burst, NET responses and bactericidal activity. (a, b) Neutrophils were labeled with OxyBURST Green H2HFF BSA in the presence of α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at MOI = 20 for 30 min and oxidative burst measured by FACS; results are expressed as MFI ±SD and repeated twice with similar results; representative experiment is shown. (c) PMA-stimulated neutrophils (5 × 105 cells) were pretreated with α-Sig-9(HA), α-Sig-9(Sia), or α-CD44 mAbs and exposed for 3 h to MOI = 10 of GAS (DNase mutant) that had been pretreated or not with hyaluronidase to remove HA capsule and NET production visualized by staining for DAPI (DNA, blue) + anti-myeloperoxidase/AlexaFluor488 (green); results are repeated 5 times in triplicate, representative fields at 32 × magnification is shown. (d) NET production was quantified by Quant-iT™ PicoGreen® assay for extracellular DNA; results are expressed as mean ± SD and repeated 2 times with similar results; representative experiment shown. (e) Neutrophils were pretreated with α-Sig-9(HA), α-Sig-9(Sia) or α-CD44 mAbs, infected with WT or isogenic ΔhasA GAS at multiplicity of infection (MOI) = 10 for 30 min, then cells lysed and dilutions plated on agar for enumeration of colony forming units to evaluate neutrophil killing of GAS. Data represent the mean + SD of triplicates; repeated 4 times with similar results; representative experiment shown. One-Way ANOVA with Dunnett’s multiple comparison test; P < 0.001 (***) or P < 0.05 (*).

Article Snippet: Siglec-Fc proteins were purified from culture supernatant by adsorption to protein A-Sepharose (GE Healthcare); human CD44–Fc chimera was purchased from R&D Systems. hSiglecs-Fc and CD44–Fc were treated with 25 mU and 5 mU of Arthrobacter ureafaciens sialidase (AUS) for 60 min to remove Sias, prior to elution from the protein A-Sepharose.

Techniques: Binding Assay, Activity Assay, Labeling, Infection, Mutagenesis, Staining, Picogreen Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Neutrophils Culture in Collagen Gel System

doi: 10.3389/fimmu.2022.816037

Figure Lengend Snippet: The morphology of Neu in 2D and 3D culture systems. (A) Morphology of Cellmatrix Type I, I-PC collagen and I-AC and Collagen type I in a static state or vibrational under light microscope. (B) Toxicity testing was evaluated by the MTT assay. Mouse bone marrow-derived Neu were embedded in Cellmatrix Type I and cultured for 24 h. MTT reagent was used to detect the survival rate of the cells. (C) The morphology of mouse peripheral blood Neu representative light microscopy photographs (600x). (D) Representative fluorescence micrograph of human peripheral blood Neu dyed by SYTO 13 (stain DNA in both live and dead eukaryotic cells) during 6 days in 2D and 3D culture systems with or without the inhibition of apoptosis z-vad-fmk. Error bars represent mean ± S.D. n = 3, ns, not statistically significantly different by One-way ANOVA.

Article Snippet: Mouse peripheral blood Neu were isolated from peripheral blood by density gradient centrifugation (Beijing Solarbio, P9201).

Techniques: Light Microscopy, MTT Assay, Derivative Assay, Cell Culture, Fluorescence, Staining, Inhibition

Journal: Frontiers in Immunology

Article Title: Neutrophils Culture in Collagen Gel System

doi: 10.3389/fimmu.2022.816037

Figure Lengend Snippet: The evaluation of function of Neu during 6 days in 2D and 3D culture system. (A) Flow cytometry analysis of ROS produced by mouse peripheral blood Neu cultured in 2D and 3D systems and corresponding statistical results (B) . Chemotactic ability of mouse peripheral blood Neu shown as the number of cells per high power field on the Petri dish bottom in 2D culture and 3D culture systems for control (randomly migrating) and WKYMVm (100 nM) stimulated mouse peripheral blood Neu (C) or control and fMLF (1 μM) stimulated human peripheral blood Neu (D) . (E) Human Neu released NETs marked by NE (Alexa Fluor 594 dye, red) and nuclear (SYTO 13, green) when stimulated by 20 μM PMA for 4 h in 2D and 3D culture systems under a confocal microscope in vitro . (F) A significantly different dsDNA release was detected using fluorescent-based Picogreen assay. (G) Meanwhile, a significantly different release of neutrophil elastase coupled dsDNA was measured using fluorescent-based Picogreen assay coated with NE antibodies. Error bars represent mean ± S.D. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not statistically significantly different by Two way ANOVA.

Article Snippet: Mouse peripheral blood Neu were isolated from peripheral blood by density gradient centrifugation (Beijing Solarbio, P9201).

Techniques: Flow Cytometry, Produced, Cell Culture, Control, Microscopy, In Vitro, Picogreen Assay

Journal: Frontiers in Immunology

Article Title: Neutrophils Culture in Collagen Gel System

doi: 10.3389/fimmu.2022.816037

Figure Lengend Snippet: The activity of Neu in 2D and 3D culture systems. (A) Survival rate was evaluated by the MTT assay. (B) Percentage of the absorbance value relative to the cells on day 1 was used to indicate the active cells. (C) DNA integrity was determined by electrophoresis of DNA isolated from cultured mouse Neu for 1-6 days. Each slot was loaded with 50 μg of genomic DNA (corresponding to 5x10 5 Neu used for preparation). Percentage of PI positive in the total mouse (D) and human (E) peripheral blood Neu which were dyed by SYTO 13 during 6 days in 2D and 3D culture systems with or without the inhibition of apoptosis z-vad-fmk was used to indicate the dead cells. Data from one of three experiments performed are shown. Error bars represent mean ± S.D. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not statistically significantly different by Two way ANOVA.

Article Snippet: Mouse peripheral blood Neu were isolated from peripheral blood by density gradient centrifugation (Beijing Solarbio, P9201).

Techniques: Activity Assay, MTT Assay, Electrophoresis, Isolation, Cell Culture, Inhibition

Journal: Frontiers in Immunology

Article Title: Neutrophils Culture in Collagen Gel System

doi: 10.3389/fimmu.2022.816037

Figure Lengend Snippet: Peripheral blood Neu co-cultured with parenchymal cells in 2D and 3D culture systems. (A) The formation of NETs (white arrow) of human peripheral blood Neu co-culture with NCM460 were observed under a confocal microscope. LY6G, PE (red) and nuclear, SYTO 13 (green). (B) The formation of NETs (white arrow) of Neu and blebbing (white arrowhead) of C2C12 were observed under a confocal microscope. Tublin, Alexa Fluor 594 dye (red) and nuclear, sytox green (green). (C) The formation of NETs (white box) produced by Neu in LPS-treated mouse was observed under a two-photon confocal microscope (APC-750-LY6G, red and nuclear-sytox green, green) in vivo . Quantification of NETs formation in the plasma was assessed by dsDNA (D) and neutrophil elastase coupled dsDNA (E) . Error bars represent mean ± S.D. n = 3, *** p < 0.001,**** p < 0.0001 by Two-way ANOVA. Each experiment was repeated three times.

Article Snippet: Mouse peripheral blood Neu were isolated from peripheral blood by density gradient centrifugation (Beijing Solarbio, P9201).

Techniques: Cell Culture, Co-Culture Assay, Microscopy, Produced, In Vivo, Clinical Proteomics

Journal: bioRxiv

Article Title: SUMM4 complex couples insulator function and DNA replication timing control

doi: 10.1101/2021.10.02.462895

Figure Lengend Snippet: a , Recombinant SUMM4. Mod(Mdg4)-His6, 67.2 (p100, cyan arrowhead) and 59.1 (p75, green arrowhead) splice forms were co-expressed with FLAG-SUUR (red arrowheads, p130 and p65) or separately in Sf9 cells and purified by FLAG or Ni-NTA affinity chromatography. Mod(Mdg4)-67.2 forms a specific complex with SUUR. b , ATPase activities of recombinant FLAG-ISWI (green bars), FLAG-SUUR (red bars) and SUMM4 (FLAG-SUUR + Mod(Mdg4)-67.2-His6, purple bars). Equimolar proteins were analyzed in reactions in the absence or presence of plasmid DNA or equivalent amounts of reconstituted oligonucleosomes, ±H1. rSUUR(KA) and rMMD4, ATPases activities of K59A mutant of SUUR (gray bars) and Mod(Mdg4)-67.2-His6 (cyan bars). Hydrolysis rates were converted to moles ATP per mole protein per minute. All reactions were performed in triplicate, error bars represent standard deviations. p -values for statistically significant differences are indicated (Mann-Whitney test). c , DNA- and nucleosome-dependent stimulation or inhibition of ATPase. The activities were analyzed as in ( b ). Statistically significant differences are shown (Mann-Whitney test). d , Nucleosome sliding activities by EpiDyne ® -PicoGreen™ assay (see Methods ) with 5 nM of recombinant ISWI, SUUR or SUMM4. Reaction time courses are shown for terminally (6-N-66) and centrally (50-N-66) positioned mononucleosomes ( Extended Data Figs. E2g - j ). RFU, relative fluorescence units produced by PicoGreen fluorescence.

Article Snippet: In brief, a recombinant ATPase over a concentration range ( Extended Data Figs. E2g - j ) was mixed with 10 nM EpiDyne biotinylated nucleosome remodeling substrate (EpiCypher) , terminally positioned 6-N-66 (219 bp DNA fragment) or centrally positioned 50-N-66 (263 bp) and 1 mM ATP in 20 µL remodeling buffer, 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl2, 0.01% (v/v) Tween-20, 0.01% (w/v) BSA.

Techniques: Recombinant, Purification, Affinity Chromatography, Plasmid Preparation, Mutagenesis, MANN-WHITNEY, Inhibition, Picogreen Assay, Fluorescence, Produced

Journal: bioRxiv

Article Title: SUMM4 complex couples insulator function and DNA replication timing control

doi: 10.1101/2021.10.02.462895

Figure Lengend Snippet: a , Physical interactions of recombinant EGG, SUUR and WDE. Untagged EGG (green arrowhead) was co-expressed with FLAG-SUUR (red arrowheads, p130 and p65) or WDE-FLAG (purple arrowhead) in Sf9 cells and purified by FLAG affinity chromatography. EGG forms a specific complex with WDE but not SUUR. b , Recombinant FLAG-SUUR(K59A) and FLAG-ISWI expressed in Sf9 cells. See legend to . c , Protein composition of in vitro reconstituted chromatin. Oligonucleosomes prepared from plasmid DNA and core histones with (+H1) or without H1 (–H1) were analyzed by SDS-PAGE and Coomassie staining. Positions of BSA, H1 and core histone bands are indicated on the right; molecular mass markers (kDa) are shown on the left. d , Micrococcal nuclease (MNase) analysis of reconstituted chromatin. Partial digestion with five different dilutions of MNase was performed on H1-free (– H1) and H1-containing (+H1) oligonucleosomes. Deproteinated DNA fragments were analyzed by agarose gel electrophoresis and stained with ethidium. Note the increased nucleosome repeat length in (+H1) lanes consistent with H1 incorporation. Triangles at the top indicate increasing MNase concentrations; 123 bp ladder was used as a molecular mass marker. e , Chromatosome stop assay. Oligonucleosomes assembled with or without H1 were subjected to partial MNase digestion, and DNA was analyzed by agarose gel electrophoresis and ethidium bromide staining. Positions of the core particle and chromatosome DNA are indicated by arrowheads. DNA fragment sizes in the 20-bp DNA ladder marker are shown. f , EpiCypher ® EpiDyne ® - PicoGreen™ assay design. EpiDyne nucleosomes encompass a restriction site shielded by the initial nucleosome position but exposed for Dpn II cleavage upon remodeling (sliding or displacement). Biotinylated substrates are immobilized on streptavidin magnetic beads. Digest by Dpn II releases the substrates from beads, and supernatant is quantified by PicoGreen™ (dsDNA detection reagent) fluorescence. g , Titration of Drosophila ISWI remodeling activity using terminally (6-N-66) or centrally (50-N-66) positioned mononucleosomes. Early reaction time points were separately plotted to indicate linear ranges. RFU, relative fluorescence units. h , Early remodeling rates for ISWI were calculated by linear regression analyses of data in respective linear ranges. ISWI exhibits a stronger remodeling activity with a centrally positioned nucleosome substrate. i , Titration of human BRG1 remodeling activity. Data are presented as in ( g ). j , Early remodeling rates for BRG1 were calculated and plotted as in ( h ). BRG1 does not exhibit a bias towards remodeling centrally or terminally positioned nucleosomes.

Article Snippet: In brief, a recombinant ATPase over a concentration range ( Extended Data Figs. E2g - j ) was mixed with 10 nM EpiDyne biotinylated nucleosome remodeling substrate (EpiCypher) , terminally positioned 6-N-66 (219 bp DNA fragment) or centrally positioned 50-N-66 (263 bp) and 1 mM ATP in 20 µL remodeling buffer, 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 3 mM MgCl2, 0.01% (v/v) Tween-20, 0.01% (w/v) BSA.

Techniques: Recombinant, Purification, Affinity Chromatography, In Vitro, Plasmid Preparation, SDS Page, Staining, Agarose Gel Electrophoresis, Marker, Picogreen Assay, Magnetic Beads, Fluorescence, Titration, Activity Assay

Journal: bioRxiv

Article Title: Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

doi: 10.1101/2025.08.26.672476

Figure Lengend Snippet: Downstream Processing (DSP) Scalability; Scaled DSP for MSC derived EVs and orthogonal analytical assessment of particle recovery and impurity removal. (a) Schematic representation of the scaled DSP workflow comprising clarification, tangential flow filtration (TFF), ion exchange (IEX) chromatography, post-IEX buffer exchange via TFF, and sterile filtration. Particle recoveries for each unit operation were determined by (b) -NTA, (c) Fl-NTA, and (d) Fl-FC. Protein removal throughout the entire DSP was quantified using (e) BCA assay, while impurity reduction was evaluated by (f) analytical chromatography through analysis of impurity-associated peak area under the curve (AUC). DNA removal was assessed via (g) PicoGreen™ assay. Representative chromatograms of analytical chromatography for DSP steps are shown in (j) , with EV-size peaks (9–14 min) highlighted in red, impurity peaks (14–20 min) in green, and late-eluting impurities (>20 min) in yellow. Final MSC-EVs preparation characterization included (h) scattering and (i) fluorescence NTA size distribution profiles, (k) transmission electron microscopy (TEM) imaging showing characteristic EV cup-shaped morphology, scale bar: 1 µm, and (l) Simple Western confirming EV marker expression (CD81, CD73, Syntenin) and absence of negative markers (calnexin, albumin). (m–o) Fibroblast scratch migration assay demonstrating retained bioactivity of purified MSC-EVs, quantified by cell density recovery within the wound area over 48 hour, supported by qualitative representative images, scale bar: Unless otherwise specified, Multicomparison statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparison test (*p<0.05, **p<0.01, ***p<0.001, ****p < 0.0001; ns not significant).

Article Snippet: Adult human dermal fibroblasts (hDFA, ATCC) were expanded according to the supplier’s instructions.

Techniques: Derivative Assay, Clarification Assay, Filtration, Chromatography, Buffer Exchange, Sterility, BIA-KA, Picogreen Assay, Fluorescence, Transmission Assay, Electron Microscopy, Imaging, Simple Western, Marker, Expressing, Migration, Purification, Comparison