Journal: Journal of the American Society of Nephrology : JASN

Article Title: C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles

doi: 10.1681/ASN.2016060637

Figure Lengend Snippet: EMVs in vasculitis plasma express B1R. Plasma samples from patients with vasculitis (n=12, patients 1–7 and 9–13 in Table 1) and healthy controls (n=15) exhibited significantly higher levels of EMVs, positive for CD105 and/or CD144. Circulating EMVs were B1R-positive and samples from patients with vasculitis had significantly higher levels of B1R compared with controls. Samples were run using a BD FACSCanto Cytometer. ***P<0.001.

Article Snippet: Flow cytometry was performed using a BD FACSCanto Cytometer and FACSDiva Software version 6.0 (Becton Dickinson Immunocytometry Systems, San Jose, CA) or a CyFlow Cube 8 flow cytometer (Sysmex Partec, Norderstedt, Germany) with FCS Express 4 Flow Research Edition software version 4.07.0003 (De Novo Software, Glendale, CA).

Techniques: Cytometry

Journal: Journal of the American Society of Nephrology : JASN

Article Title: C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles

doi: 10.1681/ASN.2016060637

Figure Lengend Snippet: B1R is found on EMVs released after perfusion of PGECs. (A) Plasma samples from patients with vasculitis (n=6) and healthy controls (n=6) perfused over PGECs at a shear stress of 2 dynes/cm2 for 15 minutes induced the release of EMVs (CD144-positive and CD105-positive MVs). Patient plasma samples contained more EMVs than control samples before perfusion (presample). Perfused plasma samples from patients with vasculitis exhibited significantly higher levels of EMVs than controls. (B) Patient samples contained more B1R-positive EMVs before perfusion (presample). Perfused samples from patients with vasculitis had significantly higher levels of B1R-positive EMVs compared with controls (the lowest value of B1R-positive EMVs in control plasma was 0.03×106/ml). Statistical comparisons were carried out between patients and controls, but not between presamples and perfused samples. Samples were analyzed using a BD FACSCanto Cytometer. **P<0.01; *P<0.05.

Article Snippet: Flow cytometry was performed using a BD FACSCanto Cytometer and FACSDiva Software version 6.0 (Becton Dickinson Immunocytometry Systems, San Jose, CA) or a CyFlow Cube 8 flow cytometer (Sysmex Partec, Norderstedt, Germany) with FCS Express 4 Flow Research Edition software version 4.07.0003 (De Novo Software, Glendale, CA).

Techniques: Cytometry

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Multicycle growth kinetics. Vero E6 cells (12-well plates, triplicates) were infected (MOI of 0.01) with rRVFV WT (rWT) or with recombinant viruses expressing Nluc ( A and B ) or Venus ( C and D ). At the indicated 24, 48, and 72 h p.i., viral titers in culture supernatants were evaluated by plaque assay (PFU/mL), and the same rWT representation was used in A and D ( A and D ). Comparison of the replication curves of viruses expressing Nluc or Venus versus rWT at different post-infection times revealed statistically significant differences (Dunnett’s method). * P < 0.05; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001. The data represent the means ± SDs of triplicate samples. At the same time, post-infection Nluc activity was evaluated (RLU: relative light units) ( B ), and Venus expression was analyzed using a fluorescence microscope ( C ). Representative images are shown. Scale bars, 100 µm. The dotted line indicates the limit of detection (100 FFU/mL) of the assay.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Plaque Assay, Comparison, Activity Assay, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Plaque assays. Vero E6 cells (10 6 cells/well, 6-well plate format) were infected with rRVFV WT (rWT) or with recombinant viruses expressing Venus (top) or Nluc (bottom) and incubated at 37°C for 3 days. Plaques were evaluated by immunostaining with the mAb anti-N (2B1), the goat pAb anti-GFP (top), or the mAb anti-Nluc (bottom). Immunostaining of rWT was performed with the mAb anti-N (2B1) or a combination of the goat pAb anti-GFP and the mAb anti-Nluc.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Incubation, Immunostaining

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Nluc activity in tissue culture supernatant versus cell extracts and Venus expression by flow cytometry analysis. Vero E6 cells (24-well plates, triplicates) were infected (MOI of 0.01) with recombinant viruses expressing Nluc. At 48 h p.i., viral titers in culture supernatants were evaluated by plaque assay (PFU/mL). The differences between the groups were calculated using one-way ANOVA with the Tukey method. * P < 0.05; *** P < 0.0002; **** P < 0.0001 ( A ). Nluc activity in tissue culture supernatants (TCSs) and cell extracts was measured. RLU: relative light units. The differences between the groups were calculated using two-way ANOVA with the Tukey method. * P < 0.05; *** P < 0.0002; **** P < 0.0001. ( B ). The data represent the means ± SDs of triplicate samples. ( C ) Flow cytometry analysis. Vero E6 cells were infected (MOI of 0.01) with recombinant viruses expressing Venus. At 24 h p.i., the cells were harvested from the culture and stained with a mAb against N conjugated with Alexa 700. The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1) and the percentage of N +, Venus+, or Venus+/ N + is indicated.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Activity Assay, Expressing, Flow Cytometry, Infection, Recombinant, Plaque Assay, Staining, Fluorescence

Journal: Journal of Virology

Article Title: Novel replication-competent reporter-expressing Rift Valley fever viruses for molecular studies

doi: 10.1128/jvi.01782-24

Figure Lengend Snippet: Multicycle growth kinetics in insect cells. C6/36 mosquito cells (T25 flasks, triplicates) were infected (MOI of 0.01) with rRVFV WT (rWT) or with recombinant viruses expressing Nluc ( A and B ) or Venus ( C and D ). At the indicated days p.i. (3, 5, and 7), viral titers in culture supernatants were evaluated by plaque assay (PFU/mL), and the same rWT representation was used in panels A and C. Dunnett’s method was used to statistically compare viruses expressing Nluc or Venus versus rWT. * P < 0.05; ** P < 0.0021; *** P < 0.0002; **** P < 0.0001 ( A and C ). The data represent the means ± SDs of triplicate samples. At the same time, p.i. Nluc activity (RLU: relative light units) was measured in ( B ), and Venus expression was evaluated using a fluorescence microscope ( D ). Representative images are shown. Scale bars, 100 µm. The dotted line indicates the limit of detection (100 FFU/mL) of the assay.

Article Snippet: The cells were analyzed by flow cytometry by gating on green (Venus) and red fluorescence (mAb anti-N 2B1 Alexa 647 conjugated) ( ) using a CyFlow Cube 8 cytometer (Sysmex).

Techniques: Infection, Recombinant, Expressing, Plaque Assay, Activity Assay, Fluorescence, Microscopy