Journal: The Journal of Infectious Diseases

Article Title: Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis

doi: 10.1093/infdis/jit572

Figure Lengend Snippet: Macrophages from patients with inflammatory disease display increased growth restriction of H37Rv and increased phagsomal fusion. Human monocyte–derived macrophages (hMDMs) from patients carrying different genetic variants in NLRP3 and/or CARD8 were infected with H37Rv at multiplicity of infection of 10. A , Luminescence was evaluated as a measure of phagocytosis on day 0. At 48 hours (ie, on day 2), luminescence was evaluated again, and the fold-change in growth was calculated as the day 2 value divided by the day 0 value. The figure shows the total fold-change from both the lysate and supernatant fraction, where the fold-change of H37Rv in cells from individuals with the respective gene variant are compared to that in cells from sex-matched control individuals with no polymorphisms. B , hMDMs from patient 2 and patient 3 were seeded on glass coverslips and stained for CD63 at 48 hours after infection. The percentage of CD63-positive phagosomes was evaluated by confocal microscopy in a blinded fashion (n = 2). Cells from healthy blood donors were included as controls (n = 18).

Article Snippet: For CD63 staining, fixed cells were stained with anti-CD63 antibody (Sanquin) and Alexa 594–conjugated goat anti-mouse antibody (Invitrogen).

Techniques: Derivative Assay, Infection, Variant Assay, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Dendritic cells derived exosomes migration to spleen and induction of inflammation are regulated by CCR7

doi: 10.1038/srep42996

Figure Lengend Snippet: ( A ) The ultrastructure of exosome by transmission electron microscopy. Bar size, 100 nm. ( B ) The size distribution profile of immature and mature DC-exos by Nanosight, revealing a size peak of 113 nm in immature DC-exos and 109 nm in mature DC-exos. ( C ) The expression of exosomes negative marker, Calnexin and positive markers, Alix, CD63 and TSG101. And also, the expression of CCR7 was detected in both cell lysis and exosomes lysis. A total of 20ug protein from DCs lysis and 5 ug protein from exosomes lysis was loaded into each lane. Full-length blots can be found in and .

Article Snippet: Antibody against CD63 was purchased from Santa Cruz Biotechnology, Alix was purchased from Cell Signaling Technology and TSG101, Calnexin and CCR7 were purchased from Abcam.

Techniques: Transmission Assay, Electron Microscopy, Expressing, Marker, Lysis

Journal: Frontiers in Immunology

Article Title: Surgical Trauma in Mice Modifies the Content of Circulating Extracellular Vesicles

doi: 10.3389/fimmu.2021.824696

Figure Lengend Snippet: Characterization of circulating EVs. (A) TEM image of the isolated particles, scale bar, 500 nm. Encircled are representative spherical particles. White arrows indicate putative lipid bilayers of EVs. The dotted insert is represented in higher magnification on the right panel. (B) Representative size distribution profile of isolated particles and their concentrations estimated by NTA. However, no differences in the control vs. S6h and the control vs, S24h and S72h comparisons were detected. (C) Western blot identification of EV enriched proteins and albumin. SDS-PAGE for CD63 western blot was run under non-reducing conditions. dEV, serum depleted of EVs, S, serum.

Article Snippet: Streptavidin-coated magnetic beads (SVMS-40-10, Spherotech) were coated with the biotinylated CD63 (clone MEM-259, BioSite Flow), CD9 (clone HI9a, Biolegend), and CD81 (clone M38, BioSite Flow) antibodies as it is described elsewhere ( ).

Techniques: Isolation, Western Blot, SDS Page

Journal: Journal of Tissue Engineering

Article Title: Extracellular vesicles delivering nuclear factor I/C for hard tissue engineering: Treatment of apical periodontitis and dentin regeneration

doi: 10.1177/20417314221084095

Figure Lengend Snippet: Higher expression of EV surface marker CD63 in apical papilla of rat apical periodontitis model. (a) Schematic diagram showing observed region of apical papilla in apical periodontitis model and healthy control, and HE staining showing inflammatory cell infiltration in inflamed apical papilla ( n = 3). (b and c) Apical papilla from healthy and inflamed mandibular incisors ( n = 3) was examined using immunohistochemical staining. Expression of CD63 (EV surface marker), caspase-3, and tumor necrosis factor-a (TNF-a) could barely be detected within healthy apical papilla (left). Significant upregulation of CD63, caspase-3, and TNFa were observed in inflamed apical papilla (right). (d) Quantitative analysis of percentage of CD63/TNFa/Caspase3 positive cell was shown ( n = 3). * p < 0.05. *** p < 0.001. # p < 0.0001.

Article Snippet: Protein concentration of EVs was quantified with a BCA Protein Assay Kit (Kangwei, Beijin, China), and the EV surface markers CD9 (1:1000, DF6565, Affinity Biosciences, USA), CD63 (1:1000, DF2305, Affinity Biosciences, USA), CD81(1:1000, Zen, Chendu, China), and TSG101 (1:1000, ab125011, Abcam, UK) were analyzed by western blot analysis.

Techniques: Expressing, Marker, Staining, Immunohistochemistry

Journal: Journal of Tissue Engineering

Article Title: Extracellular vesicles delivering nuclear factor I/C for hard tissue engineering: Treatment of apical periodontitis and dentin regeneration

doi: 10.1177/20417314221084095

Figure Lengend Snippet: Identification of EVs derived from LPS-stimulated DPCs (LPS-EVs) and establishment of in vitro model. (a) CCK8 assay detected cell viability of DPCs treated LPS (0.1, 1, 10, 100 µg/mL). (b) 1 µg/mL LPS increased EVs secretion of DPCs, compared to the other groups. (c) Schematic diagram shows the four types EV collected from EV-free culture medium of DPCs with or without LPS stimulation (0.1, 1, 10 µg/mL), which were used in subsequent assays. (d) Nanoparticle tracing assay (NTA) revealed the diameter of collected EVs was approximately 100 nm. (e) Scanning electron microscopy (SEM) of cup and saucer-shaped EVs. (f) Immunofluorescence staining confirmed that PKH26-labeled EVs (red) were endocytosed by SCAPs. DAPI (blue), and F-actin (green). (g) Western blot analysis of EVs surface markers (CD9, CD63, CD81 and TSG101). (h) CCK8 assay detected the effect of Nor-EV and LPS-EVs on cell viability of SCAPs. * p < 0.05. ** p < 0.01. *** p < 0.001.# p < 0.0001.

Article Snippet: Protein concentration of EVs was quantified with a BCA Protein Assay Kit (Kangwei, Beijin, China), and the EV surface markers CD9 (1:1000, DF6565, Affinity Biosciences, USA), CD63 (1:1000, DF2305, Affinity Biosciences, USA), CD81(1:1000, Zen, Chendu, China), and TSG101 (1:1000, ab125011, Abcam, UK) were analyzed by western blot analysis.

Techniques: Derivative Assay, In Vitro, CCK-8 Assay, Electron Microscopy, Immunofluorescence, Staining, Labeling, Western Blot

Journal: Journal of Tissue Engineering

Article Title: Extracellular vesicles delivering nuclear factor I/C for hard tissue engineering: Treatment of apical periodontitis and dentin regeneration

doi: 10.1177/20417314221084095

Figure Lengend Snippet: Construction of NFIC-EV for encapsulating and delivering NFIC. (a) Schematic diagram illustrates the method of overexpressing NFIC in HEK293FT cell line to construct NFIC-encapsulated EV. (b) qRT-PCR and Western blot analysis identified transfection efficiency of NFIC in HEK293FT cell. (c and d) Overexpressing NFIC in HEK293FT cells facilitated higher NFIC protein level in EVs derived from HEK293FT cell, which were termed as “NFIC-EV” and used in subsequent assays. Herein, NFIC protein level were normalized to EVs specific markers CD63 (e) PKH26-labeled NFIC-EV were endocytosed by SCAPs. (f and g) Treatment of NFIC-EV for 3 days upregulated NFIC of SCAPs at a protein level. * p < 0.05. *** p < 0.01. # p < 0.0001.

Article Snippet: Protein concentration of EVs was quantified with a BCA Protein Assay Kit (Kangwei, Beijin, China), and the EV surface markers CD9 (1:1000, DF6565, Affinity Biosciences, USA), CD63 (1:1000, DF2305, Affinity Biosciences, USA), CD81(1:1000, Zen, Chendu, China), and TSG101 (1:1000, ab125011, Abcam, UK) were analyzed by western blot analysis.

Techniques: Construct, Quantitative RT-PCR, Western Blot, Transfection, Derivative Assay, Labeling