Journal:

Article Title: Nef-Induced Major Histocompatibility Complex Class I Down-Regulation Is Functionally Dissociated from Its Virion Incorporation, Enhancement of Viral Infectivity, and CD4 Down-Regulation

doi:

Figure Lengend Snippet: Effect of mutations in Nef protein on MHC-I down-regulation on the surfaces of HIV-1-infected cells. (A) CEM-GFP cells (105) were infected with 5 × 105 cpm of RT from each virus obtained from transfected HeLa cells, and 3 to 5 days later the cells were reacted with an RPE-labeled anti-MHC-I antibody at 4°C for 1 h. The cells were washed, fixed with 1% formaldehyde, and analyzed for fluorescence intensity by flow cytometry. (B) The level of MHC-I expression on the GFP-positive population in virus-inoculated CEM-GFP cells was evaluated as geometric mean fluorescence using CELLQuest software (Becton Dickinson).

Article Snippet: When syncytium formation was observed a few days after infection, the cells were treated with an R-phycoerythrin (RPE)-conjugated mouse anti-MHC-I monoclonal antibody (W6/32; Dako, Glostrup, Denmark) at 4°C for 1 h. The cells were washed and fixed with 1% formaldehyde, and the fluorescence intensity for GFP and MHC-I was detected by a FACSCalibur (Becton Dickinson, Mountain View, Calif.).

Techniques: Infection, Transfection, Labeling, Fluorescence, Flow Cytometry, Expressing, Software

Journal:

Article Title: Nef-Induced Major Histocompatibility Complex Class I Down-Regulation Is Functionally Dissociated from Its Virion Incorporation, Enhancement of Viral Infectivity, and CD4 Down-Regulation

doi:

Figure Lengend Snippet: Effect of mutations in the Nef protein on CD4 down-regulation on the surfaces of cells infected with VSV-G–HIV-1 pseudotyped virus. The pseudotyped viruses were prepared by cotransfection of pCMV-G and pNL43-Ude1.K1 (B), pNL43-Ude1.K1.nM1T (C), pNL43-Ude1.K1.nM20A (D), or pNL43-Ude1.K1.nM20R (E) into HeLa cells. CEM-GFP cells (105) were infected with 2.5 × 107 cpm of RT from each pseudotyped virus, and 2 days later, cells were treated at 4°C for 1 h with RPE-labeled anti-MHC-I antibody and allophycocyanin-labeled anti-CD4 antibody. The cells were then washed, fixed with 1% formaldehyde, and analyzed for fluorescence intensity for CD4 and MHC-I in the cell population expressing GFP by flow cytometry.

Article Snippet: When syncytium formation was observed a few days after infection, the cells were treated with an R-phycoerythrin (RPE)-conjugated mouse anti-MHC-I monoclonal antibody (W6/32; Dako, Glostrup, Denmark) at 4°C for 1 h. The cells were washed and fixed with 1% formaldehyde, and the fluorescence intensity for GFP and MHC-I was detected by a FACSCalibur (Becton Dickinson, Mountain View, Calif.).

Techniques: Infection, Cotransfection, Labeling, Fluorescence, Expressing, Flow Cytometry

Journal: Current biology : CB

Article Title: The same hippocampal CA1 population simultaneously codes temporal information over multiple timescales

doi: 10.1016/j.cub.2018.03.051

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The camera objective was then aligned to the GRIN lens and lowered until visible and focused fluorescence was observed on nVista recording software (Inscopix, Inc.).

Techniques: Virus, Plasmid Preparation, Imaging, Software

Journal: Cells

Article Title: 1,8-Cineole Affects Agonists-Induced Platelet Activation, Thrombus Formation and Haemostasis

doi: 10.3390/cells10102616

Figure Lengend Snippet: Effect of 1,8-cineole on integrin αIIbβ3-mediated outside-in signalling in human platelets. Human isolated platelets (at a density of 2x10 7 cells/mL) were incubated with a vehicle control (0) or different concentrations of 1,8-cineole for 5 min and added onto fibrinogen- (100 μg/mL) coated coverslips and allowed them to spread for 45 min. Following fixation with 0.2% ( v/v ) formyl saline followed by permeabilisation with 0.2% ( v/v ) Triton X-100, the platelets were stained with Alexa Fluor 488-conjugated phalloidin for visualisation. Platelet spreading was analysed using a 100x oil immersion lens on a Nikon A1-R confocal microscope. Ten random images of view were recorded and for each sample, random locations on the slides were analysed. The number of platelets at different stages of spreading was determined by analysing the images using ImageJ. ( A ) representative images captured at 45 min of platelet spreading in the absence and presence of different concentrations of 1,8-cineole. ( Bi ) the cumulative data showing the number of platelets adhered to fibrinogen in control and 1,8-cineole treated samples. ( Bii ), the relative percentage of adhered platelets that progressed to filopodia and full spread stages on fibrinogen at 45 min. Data represent mean ± SEM ( n = 4 individual experiments using platelets obtained from four volunteers, and for each, 10 images were used for analysis). ( C ) to determine the impact of 1,8-cineole on clot retraction, human PRP was treated with various concentrations of 1,8-cineole prior to addition of 1 U/mL thrombin and monitoring of clot retraction for 2 h. The images shown are representative of four separate experiments. The data shown were calculated by measuring the remaining clot weights after 2 h of retraction. Data represent mean ± SEM ( n = 4). The p values shown (* p < 0.05, ** p < 0.001 and *** p < 0.001) are as calculated by one-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: The level of thrombus formation was observed using a Nikon A1-R confocal microscope using 20× objective.

Techniques: Isolation, Incubation, Staining, Microscopy

Journal: Cells

Article Title: 1,8-Cineole Affects Agonists-Induced Platelet Activation, Thrombus Formation and Haemostasis

doi: 10.3390/cells10102616

Figure Lengend Snippet: Impact of 1,8-cineole on thrombus formation and haemostasis. DiOC6 (a lipophilic dye) (5 μM)-labelled human whole blood was incubated with a vehicle or different concentrations of 1,8-cineole for 5 min and perfused through microfluidic channels (Vena8 BioChips) coated with collagen (400 μg/mL). Thrombus formation was observed using a 20 × objective on a Nikon A1-R confocal microscope, with images captured every 30 s up to 10 min ( A ). Quantified data represent median fluorescence intensity of thrombi formed at 10 min in control and 1,8-cineole-treated samples as calculated using NIS elements software (Nikon) and normalised to the level of median fluorescence intensity obtained for thrombi at 10 min in the vehicle treated sample ( B ). Data represents mean ± SEM ( n = 3). The p values (* p < 0.05, and ** p < 0.01) shown are as calculated by one-way ANOVA with Dunnett’s post hoc test. ( C ) Effect of 1,8-cineole on haemostasis in mice was analysed using a tail bleeding assay. Mice ( n = 6 per group) were anaesthetised and a vehicle control [0.01% ( v/v ) ethanol] or 1,8-cineole (6.25 µM or 12.5 µM) was administered via femoral artery. After 5 minutes of incubation, 3 mm of tail tip was dissected, and the tail tip was placed in sterile PBS. The time for cessation of bleeding was measured up to 20 minutes. Data represent mean ± SEM ( n = 6). The p values shown (** p < 0.01 and *** p < 0.001) are as calculated by non-parametric Kruskal–Wallis test. ( D ) To determine whether 1,8-cineole exerts any cytotoxic effects on human platelets, human isolated platelets were exposed to a positive control, a vehicle control [0.1% ( v/v ) ethanol] or various concentrations of 1,8-cineole for 30 min and the amount of LDH released (a marker for cytotoxicity) was measured at 490 nm and 650 nm using spectrophotometry. The maximum LDH release obtained with the positive control was taken as 100% and the level of LDH release for 1,8-cineole treated samples was calculated accordingly. Data represent mean ± SEM ( n = 3). The p value shown (* p < 0.05) was calculated by one-way ANOVA with post hoc Dunnett’s test.

Article Snippet: The level of thrombus formation was observed using a Nikon A1-R confocal microscope using 20× objective.

Techniques: Incubation, Microscopy, Fluorescence, Software, Isolation, Positive Control, Marker, Spectrophotometry