phosphate buffered saline pbs solution Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher pbs
    The effect of buffer species and environmental pH on immunoglobulin G (IgG) stability against denaturation by heat and urea. A, Unfolding temperatures ( T m ) of human IgG over a range of pH conditions in various buffers (see Table 1 for list) as determined by differential scanning fluorimetry (DSF) with a heating rate of 0.5°C/30 s. Optimal thermal stability of IgG was observed at pH 7.0, with a stable range of pH 5.0 to 10.0. B, Effect of buffer species on the velocity of denaturation of IgG as determined by the rate of <t>SYPRO</t> Orange fluorescence increases at various concentrations of urea. Immunoglobulin G samples were incubated at 4°C in either phosphate-buffered saline <t>(PBS)</t> or 1,4-Piperazinediethanesulfonic acid (PIPES) buffer (both pH 7.0), with various concentrations of urea. An asterisk (*) indicates significantly different from the PBS value ( P
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs/product/Thermo Fisher
    Average 99 stars, based on 45702 article reviews
    Price from $9.99 to $1999.99
    pbs - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore d pbs
    Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and <t>α-tubulin</t> (green). DNA (blue) was stained with DAPI. <t>D-PBS,</t> instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.
    D Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1785 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/d pbs/product/Millipore
    Average 99 stars, based on 1785 article reviews
    Price from $9.99 to $1999.99
    d pbs - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore pbs
    Absence of PLVAP does not affect fenestral formation or liver angiogenesis but alters immunocomplex binding to LSEC. ( a ) Scanning electron micrographs of sinusoidal vessels in 5 wk old wild-type and Plvap −/− mice. Bars, 1 µm. ( b ) Quantification (mean ± s.e.m., n = 9, 3 field/mouse, 3 mice/genotype) of the number of fenestral openings per sieve plate, the number of sieve plates per endothelial surface area, the number of fenestrations per endothelial cell area and the average surface area of a fenestral opening. ( c ) Flow cytometric analysis of liver EC (LYVE-1 + CD144 + LSEC) and their quantification per CD45 − parenchymal cells (left) and LYVE-1 − CD144 + non-sinusoidal ECs per CD45 − parenchymal cells (right)) in 5 wk old wild-type and Plvap −/− mice. Quantitative data are mean ± s.e.m. (n = 3–4 mice/genotype). ( d ) Maximal z-stack projections (48 µm) of whole-mount stainings of E14.5 liver from wild-type and Plvap −/− mice for LYVE-1 and PLVAP. Bars, 50 µm. ( e ), Quantification of sinusoidal vessel diameters in wild-type and Plvap −/− mice (mean ± s.e.m, 30 vessels/genotype, 2 mice/genotype). ( f ) Quantification of LYVE-1 and PLVAP signal areas from single optical sections (mean ± s.e.m, 10 vessels/genotype, 2 mice/genotype). ( g – i ) Flow cytometric analyses of LSEC (Live + CD45 − LYVE-1 + CD144 + cells) 2 h after an intravenous injection of Atto-488 conjugated <t>OVA-IC</t> in 5 wk old wild-type and Plvap −/− mice. ( g ) The percentage of LSEC (from all LSEC) that have taken up OVA-IC (mean ± s.e.m, 3–4 mice/genotype). ( h ) Representative histograms of the distribution of Atto-488-labeled OVA-IC in LSEC. Control, a mouse to which vehicle only <t>(PBS)</t> is administered. i Quantification of the mean fluorescence intensities (MFI) from ( g ). Representative images are from (n = 3 mice/genotype ( a ), and n = 2 mice/genotype ( d ) and 3–4 mice/genotype ( h ). *p
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34322 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs/product/Millipore
    Average 99 stars, based on 34322 article reviews
    Price from $9.99 to $1999.99
    pbs - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher dapi
    NMI accumulates with actin, the initiation competent form of pol II and nascent transcripts at the viral <t>DNA</t> centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Triple staining using the 2G2 anti-actin antibody (green), anti-NMI antibody (red) and anti-72 kD antibody (blue) showed that NMI concentrates at the same viral DNA centers as nuclear actin during Ad5 infection. (1) A profile plot of the fluorescence signal intensity along the white line intersecting a viral DNA center in the boxed area confirmed the co-localization of nuclear actin, NMI and 72kD protein. (B) Triple staining using antibodies to NMI, 72 kD protein, pol II-phosphoserine-5 (H14) or BrdU to visualize new transcripts was performed. Magnified views of viral DNA centers as indicated in merged images are shown. (C) Superresolution SIM reconstruction images of NMI (red), 72 kD (green), and <t>DAPI</t> (blue) demarcate the close association between NMI and the replication factory periphery.
    Dapi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 155801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dapi/product/Thermo Fisher
    Average 99 stars, based on 155801 article reviews
    Price from $9.99 to $1999.99
    dapi - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pbs tween 20
    NMI accumulates with actin, the initiation competent form of pol II and nascent transcripts at the viral <t>DNA</t> centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Triple staining using the 2G2 anti-actin antibody (green), anti-NMI antibody (red) and anti-72 kD antibody (blue) showed that NMI concentrates at the same viral DNA centers as nuclear actin during Ad5 infection. (1) A profile plot of the fluorescence signal intensity along the white line intersecting a viral DNA center in the boxed area confirmed the co-localization of nuclear actin, NMI and 72kD protein. (B) Triple staining using antibodies to NMI, 72 kD protein, pol II-phosphoserine-5 (H14) or BrdU to visualize new transcripts was performed. Magnified views of viral DNA centers as indicated in merged images are shown. (C) Superresolution SIM reconstruction images of NMI (red), 72 kD (green), and <t>DAPI</t> (blue) demarcate the close association between NMI and the replication factory periphery.
    Pbs Tween 20, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1622 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs tween 20/product/Thermo Fisher
    Average 99 stars, based on 1622 article reviews
    Price from $9.99 to $1999.99
    pbs tween 20 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore phosphate buffer solution pbs
    Water contact angle (WCA) of transparent superhydrophobic (TSHB) surface after different mechanical and chemical stability tests: ( a ) initial state; ( b ) high speed water jet impingement; ( c ) drawing test ( d ) ultra-sonication; ( e ) <t>IPA</t> dip; ( f ) acetone dip; ( g ) ethanol dip; ( h ) <t>PBS</t> dip.
    Phosphate Buffer Solution Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phosphate buffer solution pbs/product/Millipore
    Average 99 stars, based on 507 article reviews
    Price from $9.99 to $1999.99
    phosphate buffer solution pbs - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher 1x pbs
    Water contact angle (WCA) of transparent superhydrophobic (TSHB) surface after different mechanical and chemical stability tests: ( a ) initial state; ( b ) high speed water jet impingement; ( c ) drawing test ( d ) ultra-sonication; ( e ) <t>IPA</t> dip; ( f ) acetone dip; ( g ) ethanol dip; ( h ) <t>PBS</t> dip.
    1x Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/1x pbs/product/Thermo Fisher
    Average 99 stars, based on 6811 article reviews
    Price from $9.99 to $1999.99
    1x pbs - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pbs solution
    Surgical procedure for short-term dual chamber treatment of cortex. (A) View of mouse skull after removal of two symmetric hemi-cranial bone flaps and dura. (B) Application of paraffin wax to create two wells filled with <t>PBS.</t> (C) View of <t>Fluoro-Ruby</t> dextran-containing
    Pbs Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2896 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs solution/product/Thermo Fisher
    Average 99 stars, based on 2896 article reviews
    Price from $9.99 to $1999.99
    pbs solution - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    94
    Becton Dickinson pbs solution
    A) Fluorescent microscope images of droplets containing bacteria in absence of IPTG (top panel) and in presence of 5 mM IPTG (bottom panel). (Scale bar: 100 μm) B) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene in bulk and in droplets after addition of 5 mM IPTG over time. (n=9) Data points were normalized against the intensity of bulk culture at 12 h. C) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene 8 h after introduction of various concentration of IPTG. Data points were normalized against the intensity after 5 mM was applied. D) Fluorescent images showing GFP expressing cell clusters per droplet at 8 h, 12 h and 24 h time point post-IPTG induction. Images were taken at 20× magnification. E) Average GFP intensity per droplet as a function of time with the external concentration of IPTG ranging from 0.5 mM to 40 mM. (n=9). F) Fluorescent images showing GFP expressing cell clusters per droplet at 12 h and 24 h time point after IPTG induction in minimal <t>M9</t> vs growth <t>LB/PBS</t> media. Images were taken at 20× magnification. G) GFP intensity per droplet as a function of time upon IPTG induction in minimal M9 vs growth LB/PBS media. H) GFP intensity per droplet as a function of time with IPTG introduced prior to encapsulation at various concentrations. Fluorescent intensity curves are compared to the condition with IPTG introduced from external aqueous environment (red line). All data points for Figure 4E, G H were normalized against the fluorescence signal measured at the first time point after 2 mM was applied externally at t=0.
    Pbs Solution, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 983 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs solution/product/Becton Dickinson
    Average 94 stars, based on 983 article reviews
    Price from $9.99 to $1999.99
    pbs solution - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    99
    Millipore mtt solution
    Effect of <t>SWCNTs</t> on the number of alive and dead cells. In adhesion and suspension fractions (A) and cell viability compare with control (B) . The number of alive/dead cells was counted due to usage of trypan blue. Cell viability was analyzed by <t>MTT</t> assay. * p
    Mtt Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9203 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mtt solution/product/Millipore
    Average 99 stars, based on 9203 article reviews
    Price from $9.99 to $1999.99
    mtt solution - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Miltenyi Biotec macs buffer
    In vivo -induced expression of antimicrobial peptide (AMP) genes in trout B cells by Aeromonas salmonicida . Healthy trout were injected intraperitoneally with PBS (Ctrl) or A. salmonicida (A.sal). The IgM + and <t>IgT</t> + B cells were <t>MACS</t> sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout at 30 h postinfection and then subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in the IgM + and IgT + B cells from healthy and infected trout were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p
    Macs Buffer, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 5809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/macs buffer/product/Miltenyi Biotec
    Average 99 stars, based on 5809 article reviews
    Price from $9.99 to $1999.99
    macs buffer - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Thermo Fisher pbs phosphate buffered saline 10x ph 7 4
    In vivo -induced expression of antimicrobial peptide (AMP) genes in trout B cells by Aeromonas salmonicida . Healthy trout were injected intraperitoneally with PBS (Ctrl) or A. salmonicida (A.sal). The IgM + and <t>IgT</t> + B cells were <t>MACS</t> sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout at 30 h postinfection and then subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in the IgM + and IgT + B cells from healthy and infected trout were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p
    Pbs Phosphate Buffered Saline 10x Ph 7 4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs phosphate buffered saline 10x ph 7 4/product/Thermo Fisher
    Average 91 stars, based on 291 article reviews
    Price from $9.99 to $1999.99
    pbs phosphate buffered saline 10x ph 7 4 - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    99
    Thermo Fisher karyomax colcemid solution
    In vivo -induced expression of antimicrobial peptide (AMP) genes in trout B cells by Aeromonas salmonicida . Healthy trout were injected intraperitoneally with PBS (Ctrl) or A. salmonicida (A.sal). The IgM + and <t>IgT</t> + B cells were <t>MACS</t> sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout at 30 h postinfection and then subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in the IgM + and IgT + B cells from healthy and infected trout were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p
    Karyomax Colcemid Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/karyomax colcemid solution/product/Thermo Fisher
    Average 99 stars, based on 315 article reviews
    Price from $9.99 to $1999.99
    karyomax colcemid solution - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher pbs 1x
    Detection of cercarial elastase in  Schistosoma mansoni  cercarial samples. (A)  Cercarial elastase AL-PHA biosensor assay.  Schistosoma mansoni  cercariae were shed from infected snails, mechanically processed to produce  S. mansoni  cercarial transformation fluid (SmCTF) samples and then lyophilised in PBS (1X). Lyophilised SmCTF samples were reconstituted in water for AL-PHA biosensor assays.  (B)  AL-PHA TEV (PhaC-112L-T-G) and elastase (PhaC-112L-E-G) biosensors were treated with either PBS or SmCTF samples. Supernatant fluorescence data (483-14 nm/530-30nm) of SmCTF treated AL-PHA biosensors were normalised against mock-treated controls (PBS) of the same biosensor batch. AL-PHA beads were analysed using flow cytometry and the geometric mean (BL1-A, 488nm/530-30nm) of SmCTF treated AL-PHA biosensor beads were normalised against PBS controls of the same biosensor batch. Error bars denote standard error of the mean, n=3 (AL-PHA batches), Student  t -test *P
    Pbs 1x, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1061 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbs 1x/product/Thermo Fisher
    Average 99 stars, based on 1061 article reviews
    Price from $9.99 to $1999.99
    pbs 1x - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher hoechst 33342
    Dynamics of Bacteriocytes and Associated Nuclei during Whitefly Embryogenesis (A) Localization of bacteriocytes and associated nuclei in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition, revealed by <t>Hoechst</t> 33342 staining of DNA. The bacterial symbionts that pack the cytoplasm of bacteriocytes are evident in the bacteriocyte periphery. bc, bacteriocyte; red arrow, bacteriocyte nucleus; green arrow, egg nucleus. (B) Bacteriocyte volume in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition. Bacteriocyte volume was calculated from the diameter, assuming that the cell is a sphere, and it varied significantly with time (ANOVA: F 4,24 = 25.06, p
    Hoechst 33342, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 54383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hoechst 33342/product/Thermo Fisher
    Average 99 stars, based on 54383 article reviews
    Price from $9.99 to $1999.99
    hoechst 33342 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Millipore bsa
    <t>NPNT</t> ( nephronectin ) is a Wnt target gene and produces aldosterone via Wnt. A , Constitutive Wnt activation (ΔN-Bcat) induces NPNT mRNA expression, whereas constitutive Wnt repression (ΔN-TCF4) decreased NPNT expression compared with vector control (n=3). B , Wnt transcriptional complex T-cell factor/lymphoid enhancer factor (TCF/LEF) activity decreased in NPNT -overexpressed and increased in NPNT -silenced samples, as measured by firefly/renilla luciferase assay (n=6; n=4). C , Wnt inhibitor LGK-974 attenuated the increase in protein-normalized aldosterone production with addition of NPNT protein, compared with negative controls <t>BSA</t> and fibronectin (n=3). Bars represent mean expression per group±SEM. Statistical analyses were conducted by Student t test. * P
    Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 42788 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa/product/Millipore
    Average 99 stars, based on 42788 article reviews
    Price from $9.99 to $1999.99
    bsa - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    The effect of buffer species and environmental pH on immunoglobulin G (IgG) stability against denaturation by heat and urea. A, Unfolding temperatures ( T m ) of human IgG over a range of pH conditions in various buffers (see Table 1 for list) as determined by differential scanning fluorimetry (DSF) with a heating rate of 0.5°C/30 s. Optimal thermal stability of IgG was observed at pH 7.0, with a stable range of pH 5.0 to 10.0. B, Effect of buffer species on the velocity of denaturation of IgG as determined by the rate of SYPRO Orange fluorescence increases at various concentrations of urea. Immunoglobulin G samples were incubated at 4°C in either phosphate-buffered saline (PBS) or 1,4-Piperazinediethanesulfonic acid (PIPES) buffer (both pH 7.0), with various concentrations of urea. An asterisk (*) indicates significantly different from the PBS value ( P

    Journal: Technology in Cancer Research & Treatment

    Article Title: Sensitive Detection of Immunoglobulin G Stability Using in Real-Time Isothermal Differential Scanning Fluorimetry: Determinants of Protein Stability for Antibody-Based Therapeutics

    doi: 10.1177/1533034617714149

    Figure Lengend Snippet: The effect of buffer species and environmental pH on immunoglobulin G (IgG) stability against denaturation by heat and urea. A, Unfolding temperatures ( T m ) of human IgG over a range of pH conditions in various buffers (see Table 1 for list) as determined by differential scanning fluorimetry (DSF) with a heating rate of 0.5°C/30 s. Optimal thermal stability of IgG was observed at pH 7.0, with a stable range of pH 5.0 to 10.0. B, Effect of buffer species on the velocity of denaturation of IgG as determined by the rate of SYPRO Orange fluorescence increases at various concentrations of urea. Immunoglobulin G samples were incubated at 4°C in either phosphate-buffered saline (PBS) or 1,4-Piperazinediethanesulfonic acid (PIPES) buffer (both pH 7.0), with various concentrations of urea. An asterisk (*) indicates significantly different from the PBS value ( P

    Article Snippet: SYPRO Orange was diluted to a 40× stock in PBS (from the 5000× stock solution supplied by LifeTechnologies) and further diluted to a final concentration of 5× in DSF and RT-iDSF experiments.

    Techniques: Fluorescence, Incubation

    Inertio-elastic focusing of bioparticles based on deformability and shape ( a ) Deformation statistics of WBCs in PBS, a low-molecular weight (357 kDa) HA solution and a high-molecular weight (1,650 kDa) HA solution. The magnitude of WBC stretching is expressed in terms of aspect ratio AR = a x /a z . Scale bar, 10 µm. The error bars indicate the s.d. in the WBC aspect ratio at each flow rate. ( b ) LEF and particle trajectory analysis images of WBCs in PBS, 357 kDa HA solution and 1,650 kDa HA solution at Q = 13 ml min −1 . ( c ) Particle trajectory analysis (PTA) images of anisotropic PEG particles in 1,650 kDa HA solution at Q = 20 ml min −1 . Dashed red lines indicate channel centerline. Scale bar, 30 µm. Measurements of lateral position z and instantaneous orientation angle θ are plotted for each PEG particle in water (blue) and in the HA solution (green).

    Journal: Nature communications

    Article Title: Inertio-elastic focusing of bioparticles in microchannels at high throughput

    doi: 10.1038/ncomms5120

    Figure Lengend Snippet: Inertio-elastic focusing of bioparticles based on deformability and shape ( a ) Deformation statistics of WBCs in PBS, a low-molecular weight (357 kDa) HA solution and a high-molecular weight (1,650 kDa) HA solution. The magnitude of WBC stretching is expressed in terms of aspect ratio AR = a x /a z . Scale bar, 10 µm. The error bars indicate the s.d. in the WBC aspect ratio at each flow rate. ( b ) LEF and particle trajectory analysis images of WBCs in PBS, 357 kDa HA solution and 1,650 kDa HA solution at Q = 13 ml min −1 . ( c ) Particle trajectory analysis (PTA) images of anisotropic PEG particles in 1,650 kDa HA solution at Q = 20 ml min −1 . Dashed red lines indicate channel centerline. Scale bar, 30 µm. Measurements of lateral position z and instantaneous orientation angle θ are plotted for each PEG particle in water (blue) and in the HA solution (green).

    Article Snippet: HA sodium salt (357 kDa (Lifecore Biomedical) and 1650 kDa (Sigma-Aldrich)) was added to water (Sigma-Aldrich) for bead suspensions or phosphate-buffered saline (PBS) solution (Life Technologies) solution for cell suspensions and prepared using a roller mixer (Stuart, Sigma-Aldrich).

    Techniques: Molecular Weight, Flow Cytometry

    Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and α-tubulin (green). DNA (blue) was stained with DAPI. D-PBS, instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.

    Journal: The Journal of Reproduction and Development

    Article Title: Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes

    doi: 10.1262/jrd.2018-056

    Figure Lengend Snippet: Localization and expression level of MAD2 in fresh mouse oocytes with blocked microtubule polymerization. (A) Immunofluorescent staining of MAD2 (red) and α-tubulin (green). DNA (blue) was stained with DAPI. D-PBS, instead of the primary antibody, was used as the negative control. The inserted panel in the middle panel of the nocodazole group indicates only MAD2 signals. Scale bar = 10 μm. (B) Western blotting and expression levels of MAD2 in oocytes treated with nocodazole. β-actin was used as a loading control.

    Article Snippet: This was followed by incubation in Alexa Fluor® 594-conjugated anti-goat IgG secondary antibody (1:500 dilution, Invitrogen) for 60 min. After washing thrice with D-PBS, the oocytes were incubated with mouse anti-α-Tubulin monoclonal antibody (1:500 dilution, Sigma) as a primary antibody for 60 min at room temperature, washed thrice with D-PBS, then incubated in fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:100 dilution, Sigma) as a secondary antibody for 1 h at room temperature.

    Techniques: Expressing, Staining, Negative Control, Western Blot

    Ultraviolet (UV) crosslinking of U-X to transfected wild-type Niemann-Pick C1 (NPC1), mutant versions of NPC1, and wild-type NPC1L1 in 10–3 cells that lack NPC1. ( A and B ) Crosslinking, fluorescent labeling, and in-gel fluorescence. On day 0, 10–3 cells were set up in a six-well plate with 2 ml medium A with 5% lipoprotein-deficient serum (LPDS) per 35-mm well as described in Materials and methods. On day 1, cells were transfected by direct addition of 1 µg DNA per dish (FuGENE HD reagent) with one of the following plasmids: pcDNA3.1 control plasmid (lanes 1, 2, 9, 10); pCMV-NPC1-Flag-TEV-StrepTactin (lanes 3, 4, 11, 12); pCMV-NPC1(P691S)-Flag-TEV-StrepTactin (lanes 5, 6); pCMV-NPC1(P202A/F203A)-Flag-TEV-StrepTactin (lanes 7, 8); pCMV-NPC1L1-Flag-TEV-StrepTactin (lanes 13, 14). On day 3, all cells were incubated (without change of media) for 1 hr with 0.3 µM U-X crosslinker in the absence (–) or presence (+) of 6 µM U18666A, after which they were exposed to UV light. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluorescence as in Figure 5 . ( C ) Cholesterol esterification. On day 0, 10–3 cells were set up in medium A with 5% fetal calf serum (FCS) at 2.5 x 10 5 cells/60-mm dish. On day 1, monolayers were washed once with Dulbecco’s phosphate-buffered saline (PBS), switched to fresh medium A with 5% LPDS (devoid of penicillin and streptomycin sulfate), and then transfected with 2 µg DNA per dish with the indicated plasmids as described above. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 10 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 10% LPDS or 10% FCS as indicated. After incubation for 3 hr at 37°C, each cell monolayer was pulse-labeled for 1 hr with 0.1 mM sodium [ 14 C]oleate (5436 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the mean of triplicate incubations with individual values shown. The cellular content of [ 14 C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS (81–91 nmol/hr/mg) or FCS (88–105 nmol/hr/mg). Inset shows immunoblot analysis of whole cell extracts (6 µg) from the indicated transfection using a 1:1000 dilution of anti-Flag and anti-β-actin. DOI: http://dx.doi.org/10.7554/eLife.12177.009

    Journal: eLife

    Article Title: Identification of NPC1 as the target of U18666A, an inhibitor of lysosomal cholesterol export and Ebola infection

    doi: 10.7554/eLife.12177

    Figure Lengend Snippet: Ultraviolet (UV) crosslinking of U-X to transfected wild-type Niemann-Pick C1 (NPC1), mutant versions of NPC1, and wild-type NPC1L1 in 10–3 cells that lack NPC1. ( A and B ) Crosslinking, fluorescent labeling, and in-gel fluorescence. On day 0, 10–3 cells were set up in a six-well plate with 2 ml medium A with 5% lipoprotein-deficient serum (LPDS) per 35-mm well as described in Materials and methods. On day 1, cells were transfected by direct addition of 1 µg DNA per dish (FuGENE HD reagent) with one of the following plasmids: pcDNA3.1 control plasmid (lanes 1, 2, 9, 10); pCMV-NPC1-Flag-TEV-StrepTactin (lanes 3, 4, 11, 12); pCMV-NPC1(P691S)-Flag-TEV-StrepTactin (lanes 5, 6); pCMV-NPC1(P202A/F203A)-Flag-TEV-StrepTactin (lanes 7, 8); pCMV-NPC1L1-Flag-TEV-StrepTactin (lanes 13, 14). On day 3, all cells were incubated (without change of media) for 1 hr with 0.3 µM U-X crosslinker in the absence (–) or presence (+) of 6 µM U18666A, after which they were exposed to UV light. Cell extracts were prepared, and the crosslinked U-X was fluorescently tagged using click chemistry, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and in-gel fluorescence as in Figure 5 . ( C ) Cholesterol esterification. On day 0, 10–3 cells were set up in medium A with 5% fetal calf serum (FCS) at 2.5 x 10 5 cells/60-mm dish. On day 1, monolayers were washed once with Dulbecco’s phosphate-buffered saline (PBS), switched to fresh medium A with 5% LPDS (devoid of penicillin and streptomycin sulfate), and then transfected with 2 µg DNA per dish with the indicated plasmids as described above. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 10 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 10% LPDS or 10% FCS as indicated. After incubation for 3 hr at 37°C, each cell monolayer was pulse-labeled for 1 hr with 0.1 mM sodium [ 14 C]oleate (5436 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [ 14 C]oleate and [ 14 C]triglycerides. Each value is the mean of triplicate incubations with individual values shown. The cellular content of [ 14 C]triglycerides in all transfected cell lines did not differ significantly in cells treated with LPDS (81–91 nmol/hr/mg) or FCS (88–105 nmol/hr/mg). Inset shows immunoblot analysis of whole cell extracts (6 µg) from the indicated transfection using a 1:1000 dilution of anti-Flag and anti-β-actin. DOI: http://dx.doi.org/10.7554/eLife.12177.009

    Article Snippet: Materials We obtained U18666A, Dulbecco’s phosphate-buffered saline (PBS), CuSO4 , Tris-HCl, NaCl, FCS, chloroquine, biotin, thiourea, and Benzonase Nuclease from Sigma-Aldrich, St. Louis, MO; [1-14 C]oleic acid (55 mCi/mmol) from Perkin Elmer, Waltham, MA; Zeocin and pcDNA3.1/Zeo(-) from Life Technologies, Grand Island, NY; and FuGENE HD from Promega, Madison, WI.

    Techniques: Transfection, Mutagenesis, Labeling, Fluorescence, Plasmid Preparation, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page

    Absence of PLVAP does not affect fenestral formation or liver angiogenesis but alters immunocomplex binding to LSEC. ( a ) Scanning electron micrographs of sinusoidal vessels in 5 wk old wild-type and Plvap −/− mice. Bars, 1 µm. ( b ) Quantification (mean ± s.e.m., n = 9, 3 field/mouse, 3 mice/genotype) of the number of fenestral openings per sieve plate, the number of sieve plates per endothelial surface area, the number of fenestrations per endothelial cell area and the average surface area of a fenestral opening. ( c ) Flow cytometric analysis of liver EC (LYVE-1 + CD144 + LSEC) and their quantification per CD45 − parenchymal cells (left) and LYVE-1 − CD144 + non-sinusoidal ECs per CD45 − parenchymal cells (right)) in 5 wk old wild-type and Plvap −/− mice. Quantitative data are mean ± s.e.m. (n = 3–4 mice/genotype). ( d ) Maximal z-stack projections (48 µm) of whole-mount stainings of E14.5 liver from wild-type and Plvap −/− mice for LYVE-1 and PLVAP. Bars, 50 µm. ( e ), Quantification of sinusoidal vessel diameters in wild-type and Plvap −/− mice (mean ± s.e.m, 30 vessels/genotype, 2 mice/genotype). ( f ) Quantification of LYVE-1 and PLVAP signal areas from single optical sections (mean ± s.e.m, 10 vessels/genotype, 2 mice/genotype). ( g – i ) Flow cytometric analyses of LSEC (Live + CD45 − LYVE-1 + CD144 + cells) 2 h after an intravenous injection of Atto-488 conjugated OVA-IC in 5 wk old wild-type and Plvap −/− mice. ( g ) The percentage of LSEC (from all LSEC) that have taken up OVA-IC (mean ± s.e.m, 3–4 mice/genotype). ( h ) Representative histograms of the distribution of Atto-488-labeled OVA-IC in LSEC. Control, a mouse to which vehicle only (PBS) is administered. i Quantification of the mean fluorescence intensities (MFI) from ( g ). Representative images are from (n = 3 mice/genotype ( a ), and n = 2 mice/genotype ( d ) and 3–4 mice/genotype ( h ). *p

    Journal: Scientific Reports

    Article Title: Fenestral diaphragms and PLVAP associations in liver sinusoidal endothelial cells are developmentally regulated

    doi: 10.1038/s41598-019-52068-x

    Figure Lengend Snippet: Absence of PLVAP does not affect fenestral formation or liver angiogenesis but alters immunocomplex binding to LSEC. ( a ) Scanning electron micrographs of sinusoidal vessels in 5 wk old wild-type and Plvap −/− mice. Bars, 1 µm. ( b ) Quantification (mean ± s.e.m., n = 9, 3 field/mouse, 3 mice/genotype) of the number of fenestral openings per sieve plate, the number of sieve plates per endothelial surface area, the number of fenestrations per endothelial cell area and the average surface area of a fenestral opening. ( c ) Flow cytometric analysis of liver EC (LYVE-1 + CD144 + LSEC) and their quantification per CD45 − parenchymal cells (left) and LYVE-1 − CD144 + non-sinusoidal ECs per CD45 − parenchymal cells (right)) in 5 wk old wild-type and Plvap −/− mice. Quantitative data are mean ± s.e.m. (n = 3–4 mice/genotype). ( d ) Maximal z-stack projections (48 µm) of whole-mount stainings of E14.5 liver from wild-type and Plvap −/− mice for LYVE-1 and PLVAP. Bars, 50 µm. ( e ), Quantification of sinusoidal vessel diameters in wild-type and Plvap −/− mice (mean ± s.e.m, 30 vessels/genotype, 2 mice/genotype). ( f ) Quantification of LYVE-1 and PLVAP signal areas from single optical sections (mean ± s.e.m, 10 vessels/genotype, 2 mice/genotype). ( g – i ) Flow cytometric analyses of LSEC (Live + CD45 − LYVE-1 + CD144 + cells) 2 h after an intravenous injection of Atto-488 conjugated OVA-IC in 5 wk old wild-type and Plvap −/− mice. ( g ) The percentage of LSEC (from all LSEC) that have taken up OVA-IC (mean ± s.e.m, 3–4 mice/genotype). ( h ) Representative histograms of the distribution of Atto-488-labeled OVA-IC in LSEC. Control, a mouse to which vehicle only (PBS) is administered. i Quantification of the mean fluorescence intensities (MFI) from ( g ). Representative images are from (n = 3 mice/genotype ( a ), and n = 2 mice/genotype ( d ) and 3–4 mice/genotype ( h ). *p

    Article Snippet: To prepare ovalbumin (OVA)-anti-OVA antibody immunocomplexes, Atto488-conjugated OVA (Sigma, #41235) or Alexa Fluor 647-conjugated OVA (Q34784, Molecular probes) (50 µl from 2 mg/ml solution in PBS) was mixed with an rabbit polyclonal anti-OVA IgG (5.4 µl from 3.7 mg/ml solution, Sigma, #C6534) for 1 h at 4 o C, and 55.4 µl of the complex was then administered per mouse.

    Techniques: Binding Assay, Mouse Assay, Flow Cytometry, Injection, Labeling, Fluorescence

    Comparison of permeabilization buffer and paraformaldehyde fixation on minimum inhibition response (solid squares), maximum inhibition response (solid triangles), and Z' factor (red circles) of Plasmodium falciparum Dd2 strain parasites at varying parasitemia and 0.3% hematocrit. Combinations of Tris-HCl buffer with fixation ( A ), Tris-HCl buffer without fixation ( B ), phosphate-buffered saline (PBS) buffer with fixation ( C ), and PBS buffer without fixation ( D ) are shown. Where a linear relationship between parasitemia and minimum inhibition response was observed, linear regression (dotted) line and r 2 values were included.

    Journal: The American Journal of Tropical Medicine and Hygiene

    Article Title: Development and Optimization of a Novel 384-Well Anti-Malarial Imaging Assay Validated for High-Throughput Screening

    doi: 10.4269/ajtmh.2012.11-0302

    Figure Lengend Snippet: Comparison of permeabilization buffer and paraformaldehyde fixation on minimum inhibition response (solid squares), maximum inhibition response (solid triangles), and Z' factor (red circles) of Plasmodium falciparum Dd2 strain parasites at varying parasitemia and 0.3% hematocrit. Combinations of Tris-HCl buffer with fixation ( A ), Tris-HCl buffer without fixation ( B ), phosphate-buffered saline (PBS) buffer with fixation ( C ), and PBS buffer without fixation ( D ) are shown. Where a linear relationship between parasitemia and minimum inhibition response was observed, linear regression (dotted) line and r 2 values were included.

    Article Snippet: Hypoxanthine, 1 M NaOH, D-sorbitol, chloroquine diphosphate salt, artemisinin, 10× phosphate buffered saline (PBS) solution, EDTA, 0.5 M solution, Tris-HCl, saponin (from Quillaja Bark), Triton X-100, Giemsa stain, paraformaldehyde, dimethylformamide, and Petri dishes were purchased from Sigma Aldrich (St. Louis, MO).

    Techniques: Inhibition

    Distribution and phenotypic characterization of antigen-specific memory Th2 cells after i.n. antigen administration. ( A ) Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3. Indicated examinations were performed at day 42. ( B ) Typical staining patterns of CD44 and KJ1 among CD4 + cells in the indicated organs are shown. ( C ) Kinetics of the ratio of KJ1 + cells among CD4 + cells in the indicated organs are shown with SD. Three independent experiments were performed with similar results ( B and C ). ( D ) Representative cell surface staining profiles on KJ1 + CD4 + memory Th2 cells (red lines) and KJ1 − CD4 + recipient CD4 T cells (black lines) at day 42; gray shaded curves indicate staining with isotype-matched control antibody. Two independent experiments were performed with similar results. ( E ) For cytokine production profiles of memory Th2 cells, CD4 + cells recovered from spleen and lung were stimulated in vitro with immobilized anti-TCRβ for 6 h. Intracellular staining profiles of IL-4, IL-5, and IL-13 are shown gated on KJ1 + memory Th2 cells. ( F ) For detection of DNA synthesis, mice were injected i.p. with BrdU during days 42–47. Cells from the indicated organs were harvested at day 43 (24 h) or day 48 (6 d). Representative staining profiles of KJ1 and BrdU among CD4 + cells are shown. ( G ) For in vivo proliferation, cells from the indicated organs were harvested at day 42 and analyzed by intracellular staining of Ki67. Representative staining profiles of Ki67 gated on KJ1 + CD4 + cells in the indicated organs are shown. Two independent experiments were performed with similar results ( F and G ). ( H ) At day 42, frozen sections of lungs obtained from mice transferred with Th2 cells stained with anti-KJ1 (blue), anti-MHC class II (red), and anti-PNAd (green) are shown. Two independent experiments were performed with similar results. ( I ) At day 42, frozen sections of lungs obtained from mice transferred with the indicated type of cells stained for anti-KJ1 (blue), anti-CD3ε (red), and anti-B220 (green) are shown. (Scale bars, 20 μm.) Three independent experiments were performed with similar results.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation

    doi: 10.1073/pnas.1512600113

    Figure Lengend Snippet: Distribution and phenotypic characterization of antigen-specific memory Th2 cells after i.n. antigen administration. ( A ) Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3. Indicated examinations were performed at day 42. ( B ) Typical staining patterns of CD44 and KJ1 among CD4 + cells in the indicated organs are shown. ( C ) Kinetics of the ratio of KJ1 + cells among CD4 + cells in the indicated organs are shown with SD. Three independent experiments were performed with similar results ( B and C ). ( D ) Representative cell surface staining profiles on KJ1 + CD4 + memory Th2 cells (red lines) and KJ1 − CD4 + recipient CD4 T cells (black lines) at day 42; gray shaded curves indicate staining with isotype-matched control antibody. Two independent experiments were performed with similar results. ( E ) For cytokine production profiles of memory Th2 cells, CD4 + cells recovered from spleen and lung were stimulated in vitro with immobilized anti-TCRβ for 6 h. Intracellular staining profiles of IL-4, IL-5, and IL-13 are shown gated on KJ1 + memory Th2 cells. ( F ) For detection of DNA synthesis, mice were injected i.p. with BrdU during days 42–47. Cells from the indicated organs were harvested at day 43 (24 h) or day 48 (6 d). Representative staining profiles of KJ1 and BrdU among CD4 + cells are shown. ( G ) For in vivo proliferation, cells from the indicated organs were harvested at day 42 and analyzed by intracellular staining of Ki67. Representative staining profiles of Ki67 gated on KJ1 + CD4 + cells in the indicated organs are shown. Two independent experiments were performed with similar results ( F and G ). ( H ) At day 42, frozen sections of lungs obtained from mice transferred with Th2 cells stained with anti-KJ1 (blue), anti-MHC class II (red), and anti-PNAd (green) are shown. Two independent experiments were performed with similar results. ( I ) At day 42, frozen sections of lungs obtained from mice transferred with the indicated type of cells stained for anti-KJ1 (blue), anti-CD3ε (red), and anti-B220 (green) are shown. (Scale bars, 20 μm.) Three independent experiments were performed with similar results.

    Article Snippet: Airway inflammation was induced by exposure to a 1% solution of OVA (grade V; Sigma-Aldrich) in PBS solution, aerosolized by using a nebulizer (Omron) for 30 min. Airway hyperreactivity was assessed by methacholine-induced (Sigma-Aldrich) airflow obstruction at 24 h after the OVA challenge by a computer-controlled small animal ventilator (SCIREQ).

    Techniques: Mouse Assay, Staining, In Vitro, DNA Synthesis, Injection, In Vivo

    Antigen-specific Th2 cells and polyclonal unprimed memory phenotype CD4 T cells preferentially accumulate into the lung of mice with preformed iBALT. ( A ) Ly5.1 mice were administered i.n. with PBS solution or LPS at days 1 and 3, and effector Th2 cells from OT-II OVA-specific αβTCR Tg mice (5 × 10 6 ; Ly5.2 + ) and CD4 + cells from unprimed mice (5 × 10 6 ; Thy1.1 + ) were transferred together i.v. at day 31 and analyzed on day 38. ( B ) Representative staining profiles of Ly5.2 and CD4 from the cells in spleen and lung are shown. ( C ) Representative staining profiles of Thy1.1 and CD44 expression on CD4 + cells are shown ( Left ) with percentages of CD44 hi cells among Thy1.1 + cells in the indicated organs ( Right ). ( D ) Protocol for the selective conditional deletion of IL-7Rα gene in memory Th2 cells in the mice with iBALT. IL-7Rα fl/fl × OT-II Tg or IL-7Rα fl/fl Cre-ERT × OT-II Tg effector Th2 cells were transferred into Ly5.1 mice and subsequently challenged i.n. with OVA on days 1 and 3. At 42 d after the cell transfer, Cre-ERT activity was induced by injection of tamoxifen for five consecutive days. After a further 3 d, mice received tamoxifen for an additional five consecutive days and tissues were analyzed on day 56. For the analysis of in vivo responses, mice were challenged i.n. with OVA on days 56 and 58 and BAL fluid and airway hyperresponsiveness were assessed on day 59.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation

    doi: 10.1073/pnas.1512600113

    Figure Lengend Snippet: Antigen-specific Th2 cells and polyclonal unprimed memory phenotype CD4 T cells preferentially accumulate into the lung of mice with preformed iBALT. ( A ) Ly5.1 mice were administered i.n. with PBS solution or LPS at days 1 and 3, and effector Th2 cells from OT-II OVA-specific αβTCR Tg mice (5 × 10 6 ; Ly5.2 + ) and CD4 + cells from unprimed mice (5 × 10 6 ; Thy1.1 + ) were transferred together i.v. at day 31 and analyzed on day 38. ( B ) Representative staining profiles of Ly5.2 and CD4 from the cells in spleen and lung are shown. ( C ) Representative staining profiles of Thy1.1 and CD44 expression on CD4 + cells are shown ( Left ) with percentages of CD44 hi cells among Thy1.1 + cells in the indicated organs ( Right ). ( D ) Protocol for the selective conditional deletion of IL-7Rα gene in memory Th2 cells in the mice with iBALT. IL-7Rα fl/fl × OT-II Tg or IL-7Rα fl/fl Cre-ERT × OT-II Tg effector Th2 cells were transferred into Ly5.1 mice and subsequently challenged i.n. with OVA on days 1 and 3. At 42 d after the cell transfer, Cre-ERT activity was induced by injection of tamoxifen for five consecutive days. After a further 3 d, mice received tamoxifen for an additional five consecutive days and tissues were analyzed on day 56. For the analysis of in vivo responses, mice were challenged i.n. with OVA on days 56 and 58 and BAL fluid and airway hyperresponsiveness were assessed on day 59.

    Article Snippet: Airway inflammation was induced by exposure to a 1% solution of OVA (grade V; Sigma-Aldrich) in PBS solution, aerosolized by using a nebulizer (Omron) for 30 min. Airway hyperreactivity was assessed by methacholine-induced (Sigma-Aldrich) airflow obstruction at 24 h after the OVA challenge by a computer-controlled small animal ventilator (SCIREQ).

    Techniques: Mouse Assay, Staining, Expressing, Activity Assay, Injection, In Vivo

    Memory Th2 cells preferentially localized in iBALT. Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3, and the indicated assays were performed at day 42. ( A ) Representative cryosections of the lungs stained with H E are depicted ( Left ), as is the absolute number of iBALT structures in the lungs ( Right ). (Scale bars, 1 mm.) Mean values with SD from at least three mice per group are shown. Two independent experiments were performed with similar results. ( B ) Representative staining profiles of CD44 and KJ1 expression on CD4 + cells from the indicated organs ( Left ) and absolute cell numbers of KJ1 + CD44 hi CD4 + T cells in the indicated organs ( Right ). Mean values with SD from five mice per group are shown. More than five independent experiments were performed with similar results. ( C ) Representative confocal micrograph of lung tissue stained with anti-KJ1 (blue), anti-MHC class II (red), and anti-CD4 (green) ( Upper Left ); anti-KJ1 (blue), anti-MHC class II (red), and anti-B220 (green) ( Upper Middle ); anti-KJ1 (blue), anti-CD3ε (red), and anti-B220 (green) ( Upper Right ); anti-KJ1 (blue), anti-MHC class II (red), and anti-CD11c (green) ( Lower Left ); anti-KJ1 (blue), anti-MHC class II (Red), and anti-VCAM1 (green) ( Lower Middle ); or anti-KJ1 (blue), anti-MHC class II (Red), and anti-CD21 (green) ( Lower Right ) are shown. (Scale bars, 40 μm.) More than three independent experiments were performed with similar results. ( D ) Morphometric analysis of KJ1 + cells localized in lymphoid areas of the lungs is shown. Means and SD calculated from analysis of three slides per mouse from four mice. Two independent experiments were performed with similar results (** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation

    doi: 10.1073/pnas.1512600113

    Figure Lengend Snippet: Memory Th2 cells preferentially localized in iBALT. Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3, and the indicated assays were performed at day 42. ( A ) Representative cryosections of the lungs stained with H E are depicted ( Left ), as is the absolute number of iBALT structures in the lungs ( Right ). (Scale bars, 1 mm.) Mean values with SD from at least three mice per group are shown. Two independent experiments were performed with similar results. ( B ) Representative staining profiles of CD44 and KJ1 expression on CD4 + cells from the indicated organs ( Left ) and absolute cell numbers of KJ1 + CD44 hi CD4 + T cells in the indicated organs ( Right ). Mean values with SD from five mice per group are shown. More than five independent experiments were performed with similar results. ( C ) Representative confocal micrograph of lung tissue stained with anti-KJ1 (blue), anti-MHC class II (red), and anti-CD4 (green) ( Upper Left ); anti-KJ1 (blue), anti-MHC class II (red), and anti-B220 (green) ( Upper Middle ); anti-KJ1 (blue), anti-CD3ε (red), and anti-B220 (green) ( Upper Right ); anti-KJ1 (blue), anti-MHC class II (red), and anti-CD11c (green) ( Lower Left ); anti-KJ1 (blue), anti-MHC class II (Red), and anti-VCAM1 (green) ( Lower Middle ); or anti-KJ1 (blue), anti-MHC class II (Red), and anti-CD21 (green) ( Lower Right ) are shown. (Scale bars, 40 μm.) More than three independent experiments were performed with similar results. ( D ) Morphometric analysis of KJ1 + cells localized in lymphoid areas of the lungs is shown. Means and SD calculated from analysis of three slides per mouse from four mice. Two independent experiments were performed with similar results (** P

    Article Snippet: Airway inflammation was induced by exposure to a 1% solution of OVA (grade V; Sigma-Aldrich) in PBS solution, aerosolized by using a nebulizer (Omron) for 30 min. Airway hyperreactivity was assessed by methacholine-induced (Sigma-Aldrich) airflow obstruction at 24 h after the OVA challenge by a computer-controlled small animal ventilator (SCIREQ).

    Techniques: Mouse Assay, Staining, Expressing

    Protocol for OVA-induced airway inflammatory responses using mice with iBALT formation and IL-7Ra expression on transferred memory Th2 cells in the lung. ( A ) Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3. Airway inflammation was induced by inhalation of OVA at day 62. Bronchoalveolar lavage fluids and airway hyperreactivity was assessed at 24 h after OVA inhalation. ( B ) Effector Th2 cells from DO11.10 Tg mice were transferred into BALB/c mice. Subsequently, mice were challenged i.n. with OVA on days 1 and 3 and assessed on days 7 (1 wk) and 28 (4 wk). Representative staining profiles of IL-7Rα expression gated on CD4 + KJ1 + cells in the lung are shown; gray shaded curves indicate staining with isotype-matched control antibody.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Thy1+IL-7+ lymphatic endothelial cells in iBALT provide a survival niche for memory T-helper cells in allergic airway inflammation

    doi: 10.1073/pnas.1512600113

    Figure Lengend Snippet: Protocol for OVA-induced airway inflammatory responses using mice with iBALT formation and IL-7Ra expression on transferred memory Th2 cells in the lung. ( A ) Effector Th2 cells from DO11.10 OVA-specific αβTCR Tg mice were transferred i.v. into BALB/c mice, which were subsequently challenged i.n. with OVA or PBS solution (control) or i.p. with OVA (control) at days 1 and 3. Airway inflammation was induced by inhalation of OVA at day 62. Bronchoalveolar lavage fluids and airway hyperreactivity was assessed at 24 h after OVA inhalation. ( B ) Effector Th2 cells from DO11.10 Tg mice were transferred into BALB/c mice. Subsequently, mice were challenged i.n. with OVA on days 1 and 3 and assessed on days 7 (1 wk) and 28 (4 wk). Representative staining profiles of IL-7Rα expression gated on CD4 + KJ1 + cells in the lung are shown; gray shaded curves indicate staining with isotype-matched control antibody.

    Article Snippet: Airway inflammation was induced by exposure to a 1% solution of OVA (grade V; Sigma-Aldrich) in PBS solution, aerosolized by using a nebulizer (Omron) for 30 min. Airway hyperreactivity was assessed by methacholine-induced (Sigma-Aldrich) airflow obstruction at 24 h after the OVA challenge by a computer-controlled small animal ventilator (SCIREQ).

    Techniques: Mouse Assay, Expressing, Staining

    NMI accumulates with actin, the initiation competent form of pol II and nascent transcripts at the viral DNA centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Triple staining using the 2G2 anti-actin antibody (green), anti-NMI antibody (red) and anti-72 kD antibody (blue) showed that NMI concentrates at the same viral DNA centers as nuclear actin during Ad5 infection. (1) A profile plot of the fluorescence signal intensity along the white line intersecting a viral DNA center in the boxed area confirmed the co-localization of nuclear actin, NMI and 72kD protein. (B) Triple staining using antibodies to NMI, 72 kD protein, pol II-phosphoserine-5 (H14) or BrdU to visualize new transcripts was performed. Magnified views of viral DNA centers as indicated in merged images are shown. (C) Superresolution SIM reconstruction images of NMI (red), 72 kD (green), and DAPI (blue) demarcate the close association between NMI and the replication factory periphery.

    Journal: Experimental cell research

    Article Title: Nuclear Actin and Myosins in Adenovirus Infection

    doi: 10.1016/j.yexcr.2015.07.025

    Figure Lengend Snippet: NMI accumulates with actin, the initiation competent form of pol II and nascent transcripts at the viral DNA centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Triple staining using the 2G2 anti-actin antibody (green), anti-NMI antibody (red) and anti-72 kD antibody (blue) showed that NMI concentrates at the same viral DNA centers as nuclear actin during Ad5 infection. (1) A profile plot of the fluorescence signal intensity along the white line intersecting a viral DNA center in the boxed area confirmed the co-localization of nuclear actin, NMI and 72kD protein. (B) Triple staining using antibodies to NMI, 72 kD protein, pol II-phosphoserine-5 (H14) or BrdU to visualize new transcripts was performed. Magnified views of viral DNA centers as indicated in merged images are shown. (C) Superresolution SIM reconstruction images of NMI (red), 72 kD (green), and DAPI (blue) demarcate the close association between NMI and the replication factory periphery.

    Article Snippet: DNA was stained with DAPI (0.07 µg/ml in PBS for 5 min) and the samples were mounted in ProLong® Antifade mounting medium (Life Technologies).

    Techniques: Infection, Staining, Fluorescence

    The initiation competent form of pol II accumulates at viral DNA centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) and imaged at 21 hpi. (A) Nascent transcripts were labeled using BrUTP in permeabilized cells. Triple staining was performed using anti-BrdU (green), anti-72 kD (blue) and anti-pol II-phosphoserine-5 (H14) (red) antibodies. (B) Pol II remains associated with the viral DNA centers in the nuclear matrix. Ad5 infected cells were pre-extracted with 0.3% Triton X-100 (TTX100) to reveal components of the nuclear matrix, fixed, and subsequently labeled with antibodies against 72 kD protein (green), pol II (red) and counter-stained with DAPI (blue).

    Journal: Experimental cell research

    Article Title: Nuclear Actin and Myosins in Adenovirus Infection

    doi: 10.1016/j.yexcr.2015.07.025

    Figure Lengend Snippet: The initiation competent form of pol II accumulates at viral DNA centers. Confocal micrographs of a control (non-infected) HeLa cell and a cell infected with Ad5 wt (1000 VP/cell) and imaged at 21 hpi. (A) Nascent transcripts were labeled using BrUTP in permeabilized cells. Triple staining was performed using anti-BrdU (green), anti-72 kD (blue) and anti-pol II-phosphoserine-5 (H14) (red) antibodies. (B) Pol II remains associated with the viral DNA centers in the nuclear matrix. Ad5 infected cells were pre-extracted with 0.3% Triton X-100 (TTX100) to reveal components of the nuclear matrix, fixed, and subsequently labeled with antibodies against 72 kD protein (green), pol II (red) and counter-stained with DAPI (blue).

    Article Snippet: DNA was stained with DAPI (0.07 µg/ml in PBS for 5 min) and the samples were mounted in ProLong® Antifade mounting medium (Life Technologies).

    Techniques: Infection, Labeling, Staining

    Redistribution of myosin V and VI following Ad5 infection. Confocal micrographs of HeLa cells infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Double staining was performed using anti-MV antibody (red) and anti-72 kD antibody (green) and counter-stained with DAPI (blue). Redistribution of nuclear myosin V to the Type B viral DNA centers is indicated by arrows. (B) Superresolution SIM reconstruction images of MV (red), 72 kD (green), and DAPI (blue) demarcate the close association between MV and the replication factory periphery. (C) Double staining was performed using anti-MVI antibody (red) and anti-72 kD antibody (green) and counter-stained with DAPI (blue). Redistribution of nuclear myosin VI adjacent to type A dots is indicated by arrowhead and localization inside Type B replication centers is marked by arrows. (D) Superresolution SIM reconstruction images of MVI (red), 72 kD (green), and DAPI (blue) show the distribution of MVI within the replication factory.

    Journal: Experimental cell research

    Article Title: Nuclear Actin and Myosins in Adenovirus Infection

    doi: 10.1016/j.yexcr.2015.07.025

    Figure Lengend Snippet: Redistribution of myosin V and VI following Ad5 infection. Confocal micrographs of HeLa cells infected with Ad5 wt (1000 VP/cell) imaged at 21 hpi. (A) Double staining was performed using anti-MV antibody (red) and anti-72 kD antibody (green) and counter-stained with DAPI (blue). Redistribution of nuclear myosin V to the Type B viral DNA centers is indicated by arrows. (B) Superresolution SIM reconstruction images of MV (red), 72 kD (green), and DAPI (blue) demarcate the close association between MV and the replication factory periphery. (C) Double staining was performed using anti-MVI antibody (red) and anti-72 kD antibody (green) and counter-stained with DAPI (blue). Redistribution of nuclear myosin VI adjacent to type A dots is indicated by arrowhead and localization inside Type B replication centers is marked by arrows. (D) Superresolution SIM reconstruction images of MVI (red), 72 kD (green), and DAPI (blue) show the distribution of MVI within the replication factory.

    Article Snippet: DNA was stained with DAPI (0.07 µg/ml in PBS for 5 min) and the samples were mounted in ProLong® Antifade mounting medium (Life Technologies).

    Techniques: Infection, Double Staining, Staining

    Water contact angle (WCA) of transparent superhydrophobic (TSHB) surface after different mechanical and chemical stability tests: ( a ) initial state; ( b ) high speed water jet impingement; ( c ) drawing test ( d ) ultra-sonication; ( e ) IPA dip; ( f ) acetone dip; ( g ) ethanol dip; ( h ) PBS dip.

    Journal: Scientific Reports

    Article Title: Facile fabrication and mechanistic understanding of a transparent reversible superhydrophobic – superhydrophilic surface

    doi: 10.1038/s41598-018-37016-5

    Figure Lengend Snippet: Water contact angle (WCA) of transparent superhydrophobic (TSHB) surface after different mechanical and chemical stability tests: ( a ) initial state; ( b ) high speed water jet impingement; ( c ) drawing test ( d ) ultra-sonication; ( e ) IPA dip; ( f ) acetone dip; ( g ) ethanol dip; ( h ) PBS dip.

    Article Snippet: The chemical stability of the TSHB surface was tested by dipping the surface in various solvents such as isopropyl alcohol (IPA) (Fisher Scientific), acetone (Fisher Scientific), ethanol and phosphate buffer solution (PBS) (0.01 M concentration, Sigma-Aldrich) for a duration of 10 min.

    Techniques: Sonication, Indirect Immunoperoxidase Assay

    Surgical procedure for short-term dual chamber treatment of cortex. (A) View of mouse skull after removal of two symmetric hemi-cranial bone flaps and dura. (B) Application of paraffin wax to create two wells filled with PBS. (C) View of Fluoro-Ruby dextran-containing

    Journal: Methods (San Diego, Calif.)

    Article Title: Studying protein degradation pathways in vivo using a cranial window-based approach

    doi: 10.1016/j.ymeth.2010.12.032

    Figure Lengend Snippet: Surgical procedure for short-term dual chamber treatment of cortex. (A) View of mouse skull after removal of two symmetric hemi-cranial bone flaps and dura. (B) Application of paraffin wax to create two wells filled with PBS. (C) View of Fluoro-Ruby dextran-containing

    Article Snippet: A PBS solution containing dye (Fluoro-Ruby 10k MW dextran, 1%, Invitrogen or India Ink 1%) was used in one chamber and solution without dye in the other to test the integrity of the two wells ( ).

    Techniques:

    Comparison of the Raman spectra of D. discoideum growth and starvation EVs. (A) Proposed interpretation of Raman spectrum originated from vesicles released from D. discoideum in the starvation phase. This spectrum was obtained by averaging 15 different sets of vesicles, with a total accumulation time of 14 minutes, then subtraction of the PBS contribution, and smoothing by 2 adjacent pixels. (B) Variability of Raman spectra of D. discoideum vesicles during growth (a, b) as compared to starvation phase (c, d). Within the same phase, one can qualitatively distinguish at least 2 different species: during growth, species (b) is characterised by an increased amount of lipids as compared to species (a), and during starvation, species (d) contains an increased amount of nucleic acids and carotenoids as compared to species (c). The prominent spectral differences are highlighted by dashed oval curves.

    Journal: Journal of Extracellular Vesicles

    Article Title: Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

    doi: 10.3402/jev.v1i0.19179

    Figure Lengend Snippet: Comparison of the Raman spectra of D. discoideum growth and starvation EVs. (A) Proposed interpretation of Raman spectrum originated from vesicles released from D. discoideum in the starvation phase. This spectrum was obtained by averaging 15 different sets of vesicles, with a total accumulation time of 14 minutes, then subtraction of the PBS contribution, and smoothing by 2 adjacent pixels. (B) Variability of Raman spectra of D. discoideum vesicles during growth (a, b) as compared to starvation phase (c, d). Within the same phase, one can qualitatively distinguish at least 2 different species: during growth, species (b) is characterised by an increased amount of lipids as compared to species (a), and during starvation, species (d) contains an increased amount of nucleic acids and carotenoids as compared to species (c). The prominent spectral differences are highlighted by dashed oval curves.

    Article Snippet: EVs, present in the 12,000×g pellet, are resuspended in a phosphate-buffered solution (PBS) [pH 7.4 without calcium and magnesium (Gibco)], with a 20 to 100 volume concentration factor (X), relative to the volume of the 2,000×g supernatant.

    Techniques:

    Size distribution and concentration of D. discoideum EVs samples ( Table I ), as measured by NTA. EVs were prepared from growth medium after 48 hours of cell growth, either with the usual conditions for the 12,000×g centrifugation (in Eppendorf tubes) (a), or with the 12,000×g centrifugation performed in 30 ml tubes (see Materials and methods ) (b). The curve (c) corresponds to EVs remaining in the 12,000×g supernatant, which relates to sample (b) EVs. The 12,000×g pellets (a and b) are concentrated in PBS by factors of 20 and 100, respectively, whereas the 12,000×g supernatant (c) is as obtained after centrifugation.

    Journal: Journal of Extracellular Vesicles

    Article Title: Fast characterisation of cell-derived extracellular vesicles by nanoparticles tracking analysis, cryo-electron microscopy, and Raman tweezers microspectroscopy

    doi: 10.3402/jev.v1i0.19179

    Figure Lengend Snippet: Size distribution and concentration of D. discoideum EVs samples ( Table I ), as measured by NTA. EVs were prepared from growth medium after 48 hours of cell growth, either with the usual conditions for the 12,000×g centrifugation (in Eppendorf tubes) (a), or with the 12,000×g centrifugation performed in 30 ml tubes (see Materials and methods ) (b). The curve (c) corresponds to EVs remaining in the 12,000×g supernatant, which relates to sample (b) EVs. The 12,000×g pellets (a and b) are concentrated in PBS by factors of 20 and 100, respectively, whereas the 12,000×g supernatant (c) is as obtained after centrifugation.

    Article Snippet: EVs, present in the 12,000×g pellet, are resuspended in a phosphate-buffered solution (PBS) [pH 7.4 without calcium and magnesium (Gibco)], with a 20 to 100 volume concentration factor (X), relative to the volume of the 2,000×g supernatant.

    Techniques: Concentration Assay, Centrifugation

    A) Fluorescent microscope images of droplets containing bacteria in absence of IPTG (top panel) and in presence of 5 mM IPTG (bottom panel). (Scale bar: 100 μm) B) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene in bulk and in droplets after addition of 5 mM IPTG over time. (n=9) Data points were normalized against the intensity of bulk culture at 12 h. C) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene 8 h after introduction of various concentration of IPTG. Data points were normalized against the intensity after 5 mM was applied. D) Fluorescent images showing GFP expressing cell clusters per droplet at 8 h, 12 h and 24 h time point post-IPTG induction. Images were taken at 20× magnification. E) Average GFP intensity per droplet as a function of time with the external concentration of IPTG ranging from 0.5 mM to 40 mM. (n=9). F) Fluorescent images showing GFP expressing cell clusters per droplet at 12 h and 24 h time point after IPTG induction in minimal M9 vs growth LB/PBS media. Images were taken at 20× magnification. G) GFP intensity per droplet as a function of time upon IPTG induction in minimal M9 vs growth LB/PBS media. H) GFP intensity per droplet as a function of time with IPTG introduced prior to encapsulation at various concentrations. Fluorescent intensity curves are compared to the condition with IPTG introduced from external aqueous environment (red line). All data points for Figure 4E, G H were normalized against the fluorescence signal measured at the first time point after 2 mM was applied externally at t=0.

    Journal: Nanoscale

    Article Title: High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets

    doi: 10.1039/c6nr08224f

    Figure Lengend Snippet: A) Fluorescent microscope images of droplets containing bacteria in absence of IPTG (top panel) and in presence of 5 mM IPTG (bottom panel). (Scale bar: 100 μm) B) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene in bulk and in droplets after addition of 5 mM IPTG over time. (n=9) Data points were normalized against the intensity of bulk culture at 12 h. C) Relative fluorescent intensities of bacteria with pET vector controlling a gfp gene 8 h after introduction of various concentration of IPTG. Data points were normalized against the intensity after 5 mM was applied. D) Fluorescent images showing GFP expressing cell clusters per droplet at 8 h, 12 h and 24 h time point post-IPTG induction. Images were taken at 20× magnification. E) Average GFP intensity per droplet as a function of time with the external concentration of IPTG ranging from 0.5 mM to 40 mM. (n=9). F) Fluorescent images showing GFP expressing cell clusters per droplet at 12 h and 24 h time point after IPTG induction in minimal M9 vs growth LB/PBS media. Images were taken at 20× magnification. G) GFP intensity per droplet as a function of time upon IPTG induction in minimal M9 vs growth LB/PBS media. H) GFP intensity per droplet as a function of time with IPTG introduced prior to encapsulation at various concentrations. Fluorescent intensity curves are compared to the condition with IPTG introduced from external aqueous environment (red line). All data points for Figure 4E, G H were normalized against the fluorescence signal measured at the first time point after 2 mM was applied externally at t=0.

    Article Snippet: Equal number of droplets was then suspended in PBS solution or M9 growth medium for comparison of cell growth over time by flow cytometry (FACSCanto II, BD Biosciences, Franklin Lakes, NJ).

    Techniques: Microscopy, Positron Emission Tomography, Plasmid Preparation, Concentration Assay, Expressing, Fluorescence

    Effect of SWCNTs on the number of alive and dead cells. In adhesion and suspension fractions (A) and cell viability compare with control (B) . The number of alive/dead cells was counted due to usage of trypan blue. Cell viability was analyzed by MTT assay. * p

    Journal: Nanoscale Research Letters

    Article Title: Effect of single-walled carbon nanotubes on tumor cells viability and formation of multicellular tumor spheroids

    doi: 10.1186/s11671-015-0858-7

    Figure Lengend Snippet: Effect of SWCNTs on the number of alive and dead cells. In adhesion and suspension fractions (A) and cell viability compare with control (B) . The number of alive/dead cells was counted due to usage of trypan blue. Cell viability was analyzed by MTT assay. * p

    Article Snippet: MCF-7 cells, 1 · 104 , were seeded in a 96-well plate and incubated with SWCNTs 24 h. After that, we added 20 μl MTT solution (5 mg/ml PBS, Sigma-Aldrich, St. Louis, MO, USA) to 100 μl of cells suspension.

    Techniques: MTT Assay

    In vivo -induced expression of antimicrobial peptide (AMP) genes in trout B cells by Aeromonas salmonicida . Healthy trout were injected intraperitoneally with PBS (Ctrl) or A. salmonicida (A.sal). The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout at 30 h postinfection and then subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in the IgM + and IgT + B cells from healthy and infected trout were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p

    Journal: Frontiers in Immunology

    Article Title: B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

    doi: 10.3389/fimmu.2017.00388

    Figure Lengend Snippet: In vivo -induced expression of antimicrobial peptide (AMP) genes in trout B cells by Aeromonas salmonicida . Healthy trout were injected intraperitoneally with PBS (Ctrl) or A. salmonicida (A.sal). The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout at 30 h postinfection and then subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in the IgM + and IgT + B cells from healthy and infected trout were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p

    Article Snippet: Briefly, PBLs or HKLs were blocked with 5% fetal bovine serum (Gibco) at 4°C for 15 min, then cell suspensions were stained with mouse anti-trout IgM or anti-trout IgT mAb (1 µg/ml each) at 4°C for 30 min, washed with MACS buffer (2 mM EDTA and 0.5% BSA in PBS), and incubated at 4°C for 15 min with anti-mouse IgG magnetic beads (Miltenyi Biotec).

    Techniques: In Vivo, Expressing, Injection, Magnetic Cell Separation, Isolation, Infection, Real-time Polymerase Chain Reaction

    Cathelicidin peptides enhance the intracellular bactericidal and reactive oxygen species (ROS) activities of trout B cells . (A) Cathelicidin peptides can enhance the intracellular bactericidal activities of trout B cells. Peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout were incubated with Escherichia coli for 4 h at 17°C and then stained with mouse anti-trout IgM or anti-trout IgT mAb. The IgM + and IgT + B cells were MACS sorted and incubated with CATH-1a or CATH-2a (2 µM) for 3 h at 17°C. Non-stimulation controls (Ctrl) were included, with PBS instead of peptide. After incubation, cells were washed, lysed, and plated onto LB agar plates. Viable bacteria were counted after incubation at 37°C overnight, and results are presented as percentage of the number of bacteria for stimulation group/control group. The survival rate of the control group of IgM + B cells was set as 100%. (B) Cathelicidin peptides can enhance the intracellular ROS activities of trout B cells. Above MACS-sorted IgM + and IgT + B cells from PBLs and HKLs of trout were incubated with CATH-1a or CATH-2a (2 µM) for 3 h at 17°C. Non-stimulation controls (Ctrl) were included, with PBS instead of peptide. After incubation, cells were stained with 2,7-dichlorofluorescin diacetate for 1 h at 17°C. The fluorescence intensity of the cells was read using a fluorescence microplate reader, and the relative ROS activity was calculated. The ROS activity of the control group of IgM + B cells was set as 100% (* p

    Journal: Frontiers in Immunology

    Article Title: B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

    doi: 10.3389/fimmu.2017.00388

    Figure Lengend Snippet: Cathelicidin peptides enhance the intracellular bactericidal and reactive oxygen species (ROS) activities of trout B cells . (A) Cathelicidin peptides can enhance the intracellular bactericidal activities of trout B cells. Peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout were incubated with Escherichia coli for 4 h at 17°C and then stained with mouse anti-trout IgM or anti-trout IgT mAb. The IgM + and IgT + B cells were MACS sorted and incubated with CATH-1a or CATH-2a (2 µM) for 3 h at 17°C. Non-stimulation controls (Ctrl) were included, with PBS instead of peptide. After incubation, cells were washed, lysed, and plated onto LB agar plates. Viable bacteria were counted after incubation at 37°C overnight, and results are presented as percentage of the number of bacteria for stimulation group/control group. The survival rate of the control group of IgM + B cells was set as 100%. (B) Cathelicidin peptides can enhance the intracellular ROS activities of trout B cells. Above MACS-sorted IgM + and IgT + B cells from PBLs and HKLs of trout were incubated with CATH-1a or CATH-2a (2 µM) for 3 h at 17°C. Non-stimulation controls (Ctrl) were included, with PBS instead of peptide. After incubation, cells were stained with 2,7-dichlorofluorescin diacetate for 1 h at 17°C. The fluorescence intensity of the cells was read using a fluorescence microplate reader, and the relative ROS activity was calculated. The ROS activity of the control group of IgM + B cells was set as 100% (* p

    Article Snippet: Briefly, PBLs or HKLs were blocked with 5% fetal bovine serum (Gibco) at 4°C for 15 min, then cell suspensions were stained with mouse anti-trout IgM or anti-trout IgT mAb (1 µg/ml each) at 4°C for 30 min, washed with MACS buffer (2 mM EDTA and 0.5% BSA in PBS), and incubated at 4°C for 15 min with anti-mouse IgG magnetic beads (Miltenyi Biotec).

    Techniques: Incubation, Staining, Magnetic Cell Separation, Fluorescence, Activity Assay

    Immunofluorescence detection of cathelicidin peptide CATH-2a in trout B cells . The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout. Then cells were stained with rabbit anti-trout CATH-2a or control (rabbit anti-GST) Ab (green), and with DAPI for nuclei (blue). Data are representative of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

    doi: 10.3389/fimmu.2017.00388

    Figure Lengend Snippet: Immunofluorescence detection of cathelicidin peptide CATH-2a in trout B cells . The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout. Then cells were stained with rabbit anti-trout CATH-2a or control (rabbit anti-GST) Ab (green), and with DAPI for nuclei (blue). Data are representative of two independent experiments.

    Article Snippet: Briefly, PBLs or HKLs were blocked with 5% fetal bovine serum (Gibco) at 4°C for 15 min, then cell suspensions were stained with mouse anti-trout IgM or anti-trout IgT mAb (1 µg/ml each) at 4°C for 30 min, washed with MACS buffer (2 mM EDTA and 0.5% BSA in PBS), and incubated at 4°C for 15 min with anti-mouse IgG magnetic beads (Miltenyi Biotec).

    Techniques: Immunofluorescence, Magnetic Cell Separation, Staining

    In vitro -induced expression of antimicrobial peptide (AMP) genes in trout B cells by LPS or Aeromonas salmonicida . The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout and incubated with PBS (Ctrl), LPS (25 µg/ml), or heat-killed A. salmonicida (A.sal; cell:bacteria = 1:10) for 8 h at 17°C. Then cells were collected and subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in trout IgM + and IgT + B cells under normal and challenged situations were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p

    Journal: Frontiers in Immunology

    Article Title: B Cell Functions Can Be Modulated by Antimicrobial Peptides in Rainbow Trout Oncorhynchus mykiss: Novel Insights into the Innate Nature of B Cells in Fish

    doi: 10.3389/fimmu.2017.00388

    Figure Lengend Snippet: In vitro -induced expression of antimicrobial peptide (AMP) genes in trout B cells by LPS or Aeromonas salmonicida . The IgM + and IgT + B cells were MACS sorted from peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs) of trout and incubated with PBS (Ctrl), LPS (25 µg/ml), or heat-killed A. salmonicida (A.sal; cell:bacteria = 1:10) for 8 h at 17°C. Then cells were collected and subjected to total RNA isolation and cDNA synthesis. The relative expression levels of AMP genes in trout IgM + and IgT + B cells under normal and challenged situations were determined by quantitative real-time PCR and normalized against elongation factor 1a (* p

    Article Snippet: Briefly, PBLs or HKLs were blocked with 5% fetal bovine serum (Gibco) at 4°C for 15 min, then cell suspensions were stained with mouse anti-trout IgM or anti-trout IgT mAb (1 µg/ml each) at 4°C for 30 min, washed with MACS buffer (2 mM EDTA and 0.5% BSA in PBS), and incubated at 4°C for 15 min with anti-mouse IgG magnetic beads (Miltenyi Biotec).

    Techniques: In Vitro, Expressing, Magnetic Cell Separation, Incubation, Isolation, Real-time Polymerase Chain Reaction

    Detection of cercarial elastase in  Schistosoma mansoni  cercarial samples. (A)  Cercarial elastase AL-PHA biosensor assay.  Schistosoma mansoni  cercariae were shed from infected snails, mechanically processed to produce  S. mansoni  cercarial transformation fluid (SmCTF) samples and then lyophilised in PBS (1X). Lyophilised SmCTF samples were reconstituted in water for AL-PHA biosensor assays.  (B)  AL-PHA TEV (PhaC-112L-T-G) and elastase (PhaC-112L-E-G) biosensors were treated with either PBS or SmCTF samples. Supernatant fluorescence data (483-14 nm/530-30nm) of SmCTF treated AL-PHA biosensors were normalised against mock-treated controls (PBS) of the same biosensor batch. AL-PHA beads were analysed using flow cytometry and the geometric mean (BL1-A, 488nm/530-30nm) of SmCTF treated AL-PHA biosensor beads were normalised against PBS controls of the same biosensor batch. Error bars denote standard error of the mean, n=3 (AL-PHA batches), Student  t -test *P

    Journal: bioRxiv

    Article Title: AL-PHA beads: bioplastic-based protease biosensors for global health applications

    doi: 10.1101/2020.06.18.159921

    Figure Lengend Snippet: Detection of cercarial elastase in Schistosoma mansoni cercarial samples. (A) Cercarial elastase AL-PHA biosensor assay. Schistosoma mansoni cercariae were shed from infected snails, mechanically processed to produce S. mansoni cercarial transformation fluid (SmCTF) samples and then lyophilised in PBS (1X). Lyophilised SmCTF samples were reconstituted in water for AL-PHA biosensor assays. (B) AL-PHA TEV (PhaC-112L-T-G) and elastase (PhaC-112L-E-G) biosensors were treated with either PBS or SmCTF samples. Supernatant fluorescence data (483-14 nm/530-30nm) of SmCTF treated AL-PHA biosensors were normalised against mock-treated controls (PBS) of the same biosensor batch. AL-PHA beads were analysed using flow cytometry and the geometric mean (BL1-A, 488nm/530-30nm) of SmCTF treated AL-PHA biosensor beads were normalised against PBS controls of the same biosensor batch. Error bars denote standard error of the mean, n=3 (AL-PHA batches), Student t -test *P

    Article Snippet: S. mansoni cercarial elastase AL-PHA assays: 100 μl (total volume) reactions were setup within 1.5 ml microtubes and included the following components, 1 μl of purified AL-PHA beads, 10 μl of reconstituted SmCTF sample and 89 μl PBS (1X).

    Techniques: Biosensor Assay, Infection, Transformation Assay, Fluorescence, Flow Cytometry

    Dynamics of Bacteriocytes and Associated Nuclei during Whitefly Embryogenesis (A) Localization of bacteriocytes and associated nuclei in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition, revealed by Hoechst 33342 staining of DNA. The bacterial symbionts that pack the cytoplasm of bacteriocytes are evident in the bacteriocyte periphery. bc, bacteriocyte; red arrow, bacteriocyte nucleus; green arrow, egg nucleus. (B) Bacteriocyte volume in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition. Bacteriocyte volume was calculated from the diameter, assuming that the cell is a sphere, and it varied significantly with time (ANOVA: F 4,24 = 25.06, p

    Journal: Current Biology

    Article Title: Maternal Inheritance of a Single Somatic Animal Cell Displayed by the Bacteriocyte in the Whitefly Bemisia tabaci

    doi: 10.1016/j.cub.2017.12.041

    Figure Lengend Snippet: Dynamics of Bacteriocytes and Associated Nuclei during Whitefly Embryogenesis (A) Localization of bacteriocytes and associated nuclei in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition, revealed by Hoechst 33342 staining of DNA. The bacterial symbionts that pack the cytoplasm of bacteriocytes are evident in the bacteriocyte periphery. bc, bacteriocyte; red arrow, bacteriocyte nucleus; green arrow, egg nucleus. (B) Bacteriocyte volume in eggs at 0, 4, 5–6, 7, and 8 days post-oviposition. Bacteriocyte volume was calculated from the diameter, assuming that the cell is a sphere, and it varied significantly with time (ANOVA: F 4,24 = 25.06, p

    Article Snippet: Eggs deposited at day 0 were dechorionated by 60% Clorox bleach (3.6% hypochlorite) in PBS for 5 min and then wash with PBS twice, fixed by 4% paraformaldehyde (PFA) at room temperature for 1 h, and then permeabilized with 0.1% Triton X-100 in PBS for 1.5 h. The samples were incubated with Hoechst 33342 (10 μg ml-1 in PBS, Thermo Scientific) at room temperature for 20 min. After the dechorionation treatment, the nuclei in the embryos at late embryogenesis did not stain well.

    Techniques: Staining

    NPNT ( nephronectin ) is a Wnt target gene and produces aldosterone via Wnt. A , Constitutive Wnt activation (ΔN-Bcat) induces NPNT mRNA expression, whereas constitutive Wnt repression (ΔN-TCF4) decreased NPNT expression compared with vector control (n=3). B , Wnt transcriptional complex T-cell factor/lymphoid enhancer factor (TCF/LEF) activity decreased in NPNT -overexpressed and increased in NPNT -silenced samples, as measured by firefly/renilla luciferase assay (n=6; n=4). C , Wnt inhibitor LGK-974 attenuated the increase in protein-normalized aldosterone production with addition of NPNT protein, compared with negative controls BSA and fibronectin (n=3). Bars represent mean expression per group±SEM. Statistical analyses were conducted by Student t test. * P

    Journal: Hypertension (Dallas, Tex. : 1979)

    Article Title: Physiological and Pathological Roles in Human Adrenal of the Glomeruli-Defining Matrix Protein NPNT (Nephronectin)

    doi: 10.1161/HYPERTENSIONAHA.117.09156

    Figure Lengend Snippet: NPNT ( nephronectin ) is a Wnt target gene and produces aldosterone via Wnt. A , Constitutive Wnt activation (ΔN-Bcat) induces NPNT mRNA expression, whereas constitutive Wnt repression (ΔN-TCF4) decreased NPNT expression compared with vector control (n=3). B , Wnt transcriptional complex T-cell factor/lymphoid enhancer factor (TCF/LEF) activity decreased in NPNT -overexpressed and increased in NPNT -silenced samples, as measured by firefly/renilla luciferase assay (n=6; n=4). C , Wnt inhibitor LGK-974 attenuated the increase in protein-normalized aldosterone production with addition of NPNT protein, compared with negative controls BSA and fibronectin (n=3). Bars represent mean expression per group±SEM. Statistical analyses were conducted by Student t test. * P

    Article Snippet: Proteins and Chemicals Proteins used in this study were BSA (A9576; Sigma), NPNT (4298-NP-050; R & D systems), fibronectin (FC010; Millipore), and laminin (AG56P; Millipore).

    Techniques: Activation Assay, Expressing, Plasmid Preparation, Activity Assay, Luciferase

    NPNT ( nephronectin ) is proadhesive in normal adrenal and aldosterone-producing adenoma (APA) cells, but antiadhesive in H295R. A , NPNT is proadhesive in ( i ) HEK, ( ii ) normal primary adrenal, ( iii ) zona fasciculata (ZF)–like APA, and ( iv ) zona glomerulosa (ZG)–like APA, but antiadhesive in ( v ) H295R, as demonstrated by wells precoated with PBS, BSA, NPNT, or laminin, and measured by Xcelligence cell impedance as cell index over time (4 h for cell lines, 10 h for primary adrenal cells; n=2 for ZF-like APA and ZG-like APA, n=4 for the rest). B , NPNT is antiadhesive in H295R cells, as confirmed by Hoechst fluorescent stain assay measuring % maximum fluorescence as a representation of number of cells adhered to well with increasing concentrations of BSA, NPNT , and laminin precoating (n=3 for each protein at each concentration). Bars represent mean values per group±SEM.

    Journal: Hypertension (Dallas, Tex. : 1979)

    Article Title: Physiological and Pathological Roles in Human Adrenal of the Glomeruli-Defining Matrix Protein NPNT (Nephronectin)

    doi: 10.1161/HYPERTENSIONAHA.117.09156

    Figure Lengend Snippet: NPNT ( nephronectin ) is proadhesive in normal adrenal and aldosterone-producing adenoma (APA) cells, but antiadhesive in H295R. A , NPNT is proadhesive in ( i ) HEK, ( ii ) normal primary adrenal, ( iii ) zona fasciculata (ZF)–like APA, and ( iv ) zona glomerulosa (ZG)–like APA, but antiadhesive in ( v ) H295R, as demonstrated by wells precoated with PBS, BSA, NPNT, or laminin, and measured by Xcelligence cell impedance as cell index over time (4 h for cell lines, 10 h for primary adrenal cells; n=2 for ZF-like APA and ZG-like APA, n=4 for the rest). B , NPNT is antiadhesive in H295R cells, as confirmed by Hoechst fluorescent stain assay measuring % maximum fluorescence as a representation of number of cells adhered to well with increasing concentrations of BSA, NPNT , and laminin precoating (n=3 for each protein at each concentration). Bars represent mean values per group±SEM.

    Article Snippet: Proteins and Chemicals Proteins used in this study were BSA (A9576; Sigma), NPNT (4298-NP-050; R & D systems), fibronectin (FC010; Millipore), and laminin (AG56P; Millipore).

    Techniques: Staining, Fluorescence, Concentration Assay