Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: (A) Workflow for collection of extracellular vesicles from RAW264.7 macrophages infected with L. donovani parasites. Infection was performed in media supplemented with exosome-depleted serum. After 24 h, the culture medium was removed. Cultures were washed to remove uninternalized parasites and replenished with fresh medium supplemented with exosome-depleted serum. After an additional 48 h, culture medium was recovered, pooled, and processed following the centrifugation and filtration steps shown in the figure. (B) Nanoparticle tracking analysis was performed from which particle size distribution and particle concentration was obtained. Plot of particles/cell was calculated using cell count at the end of the infection. Data for graphs were obtained from multiple experiments (ceEV n = 7, LieEV n = 7; * P = 0.0082). (C) Representative image of vesicles in LieEV preparation processed for scanning electron microscopy. (D) Representative transmission electron microscopy image of immunogold CD9-labeled particles in LieEVs. Arrows point to gold particles denoting reactivity of antibody.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Centrifugation, Filtration, Concentration Assay, Cell Counting, Electron Microscopy, Transmission Assay, Labeling

Journal: Life Science Alliance

Article Title: Leishmania -infected macrophages release extracellular vesicles that can promote lesion development

doi: 10.26508/lsa.202000742

Figure Lengend Snippet: The proteome of LieEVs recovered after 72-h infection was compared with the proteome of ceEVs from uninfected cells. (A) Venn diagram was plotted using proteins identified by mass spectrometry and partitioned according to sample type. The presence and absence of host molecules with and without infection and common to both samples are indicated. (B) The protein content of the preparations was revealed by Ponceau S staining of the blots to normalize for material loaded into each well for each sample type. Approximately 1 × 10 10 particles from EV preparations from three replicate experiments and 50 μg of lysates from infected cells at the 72 h infection point were analyzed. (C) Western blot was performed to confirm the presence of known and novel exosome markers. Uninfected macrophages were treated in an identical manner as infected samples. The blots were then probed with anti-CD9, stripped, and probed with anti-Annexin A3. Identical blots were probed initially with anti-CD63, stripped, and probed with anti-calnexin. (D) Quantification was performed by measuring the mean gray background area using ImageJ software. Background pixel density was subtracted from the inverse of each measurement to obtain relative quantification values. Analysis of the blots showed that CD9 was significantly more abundant in LieEVs than ceEVs and cell lysates (* P = 0.0261, n = 3), whereas levels of CD63 were comparable for all samples. Annexin A3 was significantly more abundant in LieEVs than in ceEVs (* P = 0.0123, n = 3). Calnexin was barely detected in EVs as compared with cell lysates. Statistical test for differences was by ANOVA. Source data are available for this figure.

Article Snippet: The HM20-embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold-labeled with an anti-CD9 antibody (1/50 dilution vol/vol) (PB9930; Boster Bio) using a previously described method ( ).

Techniques: Infection, Mass Spectrometry, Staining, Western Blot, Software, Quantitative Proteomics

Journal: bioRxiv

Article Title: Optogenetics-integrated gut organ culture system connects enteric neurons dynamics and gut homeostasis

doi: 10.1101/2024.03.28.587149

Figure Lengend Snippet: (A-B) Flow cytometry analysis of colonic epithelial layer, lamina propria, and muscularis myenteric plexus, dissected from ChAT-ChR2 (A) or wild-type (WT) (B) mice. Cells were stained using anti-CD45, SCA1 and CD9. ChAT-EYFP-expressing enteric neurons (CD45,Sca1 negative , CD9 high ) are shown in the muscularis myenteric plexus of ChAT-ChR2 mice (bottom left). (C and E) Time lapse frame sequence following a train of 100msec pulses applied for 10 secs (stimulus onset is defined as t=0) using a set of LEDs, revealing the robust increase in calcium induced by the ChR2 following blue (F) but not yellow light (C) illumination, as well as the readily observed contraction of the tissue. (D and F) The average fluorescence signal change in the selected fields of view within the tissue, revealing the robust increase in calcium fluorescence signal following blue light illumination.

Article Snippet: Purified single-cells suspensions were stained using antibodies against mouse CD45 (Miltenyi Biotec; 130-117-498), SCA1 (Miltenyi Biotec; 130-102-832) and CD9 (Miltenyi Biotec; 130-102-612) for 15min at 4°C.

Techniques: Flow Cytometry, Staining, Expressing, Sequencing, Fluorescence