phosphate buffered saline Search Results


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  • 99
    Thermo Fisher of phosphate buffered saline
    Of Phosphate Buffered Saline, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pbs
    Anti-SAP antibodies block SAP and PulA enzymatic activity. (A) COH1 strain was grown in <t>pullulan</t> and assayed for the capacity to degrade pullulan in the presence of different anti-sera. The effect of specific anti-sera was tested in a dose range between 0.5 and 2%. White circles indicate the effect of a mouse <t>anti-PBS</t> serum; black circles indicate the effect of an antiserum from an unrelated surface-associated protein; black squares indicate the effect of a mouse anti-SAP serum; white triangles indicate the effect of a mouse anti-SAP serum absorbed to a CNBR resin coated with SAP. (B) as in (A) except for testing the inhibitory activity of an anti-SAP serum on GAS SF370 strain.
    Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 34386 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbs
    Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor <t>AMD3100</t> at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either <t>PBS</t> or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.
    Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 47152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbs buffer
    Calorimetry data for the titration of <t>OD4.2.2</t> with VRC-PG04 antibody in <t>PBS</t> buffer (pH 7.4). Measurements were carried out in triplicate with a representative titration result shown. The top panel shows raw data with the area under each spike proportional to the heat produced at each injection of VRC-PG04 Fab. The lower panel shows integrated areas normalized to the number of moles of VRC-PG04 Fab.
    Pbs Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs  (Lonza)
    99
    Lonza pbs
    CAP256 gp140-FL-IP-His protein α-Env <t>ELISA.</t> Binding ELISAs using anti-Env human monoclonal antibodies to CAP256 gp140-FL-IP-His. (A) PGT128 and PGT135 binding indicate the presence of the Env V3-glycan supersite. Similarly, the CD4 binding site using VRC01 The V2-glycan epitope was detected with PG9 (A), but no binding was observed for the native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08 (B). In line with this, clear ELISA signals were observed for F105 and 446-52D, indicative of the presence of misfolded Env (A). (C) Control soluble, trimeric BG505_664-His Env protein performed as expected, with ELISA signals for native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08. (D) No binding of MAbs was observed for the no protein <t>(PBS)</t> control.
    Pbs, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 1233 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher free pbs
    Comparison of inflammatory parameters for cobalt monoxide (CoO) nanoparticles <t>(NPs)</t> and cobalt chloride (CoCl 2 ). Number of ( A ) total cells, ( B ) macrophages, ( C ) neutrophils, ( D ) eosinophils, and levels of ( E ) LDH, ( F ) total protein, ( G ) IL-6, and ( H ) eotaxin. Both treatment groups had same doses for cobalt ions (31, 79, and 315 μg/rat). Note that the inflammatory parameters of CoO NPs were similar with the CoCl 2 which implies that the toxicity of CoO NPs was derived from their solubilized cobalt ions. Values are mean ± SEM ( n = 4) for each treatment group. The data from CoO NPs were compared with the vehicle control (Ca 2+ - and Mg 2+ -free phosphate buffered saline) to determine statistical significance. * p
    Free Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 656 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sterile pbs
    Multipotent adult progenitor cells <t>(MAPC)</t> administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to <t>PBS</t> and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.
    Sterile Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sterile pbs
    High-sensitivity flow-cytometry analysis of fractionated <t>IDG</t> revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in <t>PBS-loaded</t> (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.
    Sterile Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbs  (Bio-Rad)
    99
    Bio-Rad pbs
    The implementation of size-exclusion chromatography to remove <t>Optiprep</t> remnants from EV samples. ( a ) Optiprep density gradients were loaded with <t>PBS</t> to obtain blank EV-density fractions (F1) and with CCM to obtain EV-containing fractions (F2). EVs were retrieved by pelleting at 100,000 g (P1) or by size-exclusion chromatography (S1). Quantity of Optiprep in S1 and P1 was calculated using DC Protein assay. SEC was performed on F2 and consecutive SEC fractions (S2) were analyzed by NTA and protein analysis for GFP and CD9. ( b ) Comparative graph of NTA from S2 (connected line, marked area = SD) and Optiprep quantification of S1 (three differently colored bars representing three independent gradients, SD indicated). ( c ) Protein analysis of S2 by Western blot analysis for GFP and CD9 quantification by TRIFic CD9 assay. Original immunostaining results are shown in Supplementary Fig. 10 . Abbreviations: PBS: phosphate buffered saline. CCM: concentrated conditioned medium. GFP: green fluorescent protein. EV: extracellular vesicle. WB: Western blot. NTA: Nanoparticle tracking analysis. SEC: size-exclusion chromatography.
    Pbs, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 1x pbs
    Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a)  Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS.  (b)  Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA  (c,d)  Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p
    1x Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3453 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dulbeccos pbs
    Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a)  Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS.  (b)  Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA  (c,d)  Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p
    Dulbeccos Pbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore materials phosphate buffered saline
    Validation of puromycin incorporation into nascent proteins and Restrictocin A-induced inhibition of protein synthesis during viral entry. ( A ) H1-HeLa cells were incubated with 100 µg/ml α-sarcin or 50 µg/ml Restrictocin A for 6 hr at 37˚C, followed by treatment with cycloheximide for 30 min prior to a 20 min pulse with 20 µg/ml puromycin. Protein lysates were harvested, resolved by SDS-PAGE and subjected to immunoblotting using antibodies against puromycin and actin. ( B ) H1-HeLa cells were adsorbed for 1 hr with gradient-purified eHAV or naked HAV (~1000 GEs/cell) at 37˚C or with HRV14 (10 PFU/cell) at 33˚C in presence or absence of 50 µg/ml Restrictocin A. The inoculum was removed, cells were rinsed with <t>PBS,</t> and incubated for another 5 hr. Cells were then pulsed with puromycin for 20 min and protein lysates were harvested, resolved by SDS-PAGE, and subjected to immublobotting using antibodies against puromycin and actin. Band intensities were quantified, normalized to loading control, and presented as a percentage of virus plus α-sarcin relative to virus alone. Alternatively, viruses were incubated overnight at <t>4˚C</t> with human ‘JC’ plasma containing neutralizing HAV antibodies and inoculated as such.
    Materials Phosphate Buffered Saline, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore pbs pbst
    Validation of puromycin incorporation into nascent proteins and Restrictocin A-induced inhibition of protein synthesis during viral entry. ( A ) H1-HeLa cells were incubated with 100 µg/ml α-sarcin or 50 µg/ml Restrictocin A for 6 hr at 37˚C, followed by treatment with cycloheximide for 30 min prior to a 20 min pulse with 20 µg/ml puromycin. Protein lysates were harvested, resolved by SDS-PAGE and subjected to immunoblotting using antibodies against puromycin and actin. ( B ) H1-HeLa cells were adsorbed for 1 hr with gradient-purified eHAV or naked HAV (~1000 GEs/cell) at 37˚C or with HRV14 (10 PFU/cell) at 33˚C in presence or absence of 50 µg/ml Restrictocin A. The inoculum was removed, cells were rinsed with <t>PBS,</t> and incubated for another 5 hr. Cells were then pulsed with puromycin for 20 min and protein lysates were harvested, resolved by SDS-PAGE, and subjected to immublobotting using antibodies against puromycin and actin. Band intensities were quantified, normalized to loading control, and presented as a percentage of virus plus α-sarcin relative to virus alone. Alternatively, viruses were incubated overnight at <t>4˚C</t> with human ‘JC’ plasma containing neutralizing HAV antibodies and inoculated as such.
    Pbs Pbst, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore free pbs
    Validation of puromycin incorporation into nascent proteins and Restrictocin A-induced inhibition of protein synthesis during viral entry. ( A ) H1-HeLa cells were incubated with 100 µg/ml α-sarcin or 50 µg/ml Restrictocin A for 6 hr at 37˚C, followed by treatment with cycloheximide for 30 min prior to a 20 min pulse with 20 µg/ml puromycin. Protein lysates were harvested, resolved by SDS-PAGE and subjected to immunoblotting using antibodies against puromycin and actin. ( B ) H1-HeLa cells were adsorbed for 1 hr with gradient-purified eHAV or naked HAV (~1000 GEs/cell) at 37˚C or with HRV14 (10 PFU/cell) at 33˚C in presence or absence of 50 µg/ml Restrictocin A. The inoculum was removed, cells were rinsed with <t>PBS,</t> and incubated for another 5 hr. Cells were then pulsed with puromycin for 20 min and protein lysates were harvested, resolved by SDS-PAGE, and subjected to immublobotting using antibodies against puromycin and actin. Band intensities were quantified, normalized to loading control, and presented as a percentage of virus plus α-sarcin relative to virus alone. Alternatively, viruses were incubated overnight at <t>4˚C</t> with human ‘JC’ plasma containing neutralizing HAV antibodies and inoculated as such.
    Free Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Bio-Rad pbs buffer
    Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) <t>AcOSu,</t> <t>PBS</t> (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .
    Pbs Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 96/100, based on 349 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore x pbs
    Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) <t>AcOSu,</t> <t>PBS</t> (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .
    X Pbs, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher pbs wash
    Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) <t>AcOSu,</t> <t>PBS</t> (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .
    Pbs Wash, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1666 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Anti-SAP antibodies block SAP and PulA enzymatic activity. (A) COH1 strain was grown in pullulan and assayed for the capacity to degrade pullulan in the presence of different anti-sera. The effect of specific anti-sera was tested in a dose range between 0.5 and 2%. White circles indicate the effect of a mouse anti-PBS serum; black circles indicate the effect of an antiserum from an unrelated surface-associated protein; black squares indicate the effect of a mouse anti-SAP serum; white triangles indicate the effect of a mouse anti-SAP serum absorbed to a CNBR resin coated with SAP. (B) as in (A) except for testing the inhibitory activity of an anti-SAP serum on GAS SF370 strain.

    Journal: PLoS ONE

    Article Title: Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

    doi: 10.1371/journal.pone.0003787

    Figure Lengend Snippet: Anti-SAP antibodies block SAP and PulA enzymatic activity. (A) COH1 strain was grown in pullulan and assayed for the capacity to degrade pullulan in the presence of different anti-sera. The effect of specific anti-sera was tested in a dose range between 0.5 and 2%. White circles indicate the effect of a mouse anti-PBS serum; black circles indicate the effect of an antiserum from an unrelated surface-associated protein; black squares indicate the effect of a mouse anti-SAP serum; white triangles indicate the effect of a mouse anti-SAP serum absorbed to a CNBR resin coated with SAP. (B) as in (A) except for testing the inhibitory activity of an anti-SAP serum on GAS SF370 strain.

    Article Snippet: The mixtures contained 1% (w/v) pullulan, glycogen type IX, amylose, amylopectin or soluble starch (Sigma) dissolved in PBS (pH 7.0), and appropriately diluted enzyme in a total volume of 500 µL.

    Techniques: Blocking Assay, Activity Assay

    Swelling ratio of GelMA and GelMA/PEGDA hydrogels in PBS solution at room temperature (* P

    Journal: Materials

    Article Title: Development of a Photo-Crosslinking, Biodegradable GelMA/PEGDA Hydrogel for Guided Bone Regeneration Materials

    doi: 10.3390/ma11081345

    Figure Lengend Snippet: Swelling ratio of GelMA and GelMA/PEGDA hydrogels in PBS solution at room temperature (* P

    Article Snippet: The lyophilized GelMA was sterilized by ethylene oxide, and PEGDA and I2959 were dissolved in PBS buffer and filter-sterilized through 0.22 μm filter (produced by Millipore, Burlington, MA, USA).

    Techniques:

    Pirenzepine release over 24 hours in 4 mL of PBS from contact lenses after 24 hours of uptake in 1 mg/mL pirenzepine solution. Notes: Bars represent standard deviation. ▲, etafilcon A; ◆, ocufilcon B; □, omafilcon B (MF); ○, omafilcon A (SV); Δ, omafilcon A (MF); ●, narafilcon A; ✹, nelfilcon A (SV); ◇, nelfilcon A (MF); ⋆, comfilcon A (MF); ∇, delefilcon A (n=4/ material). SV and MF denote the single vision and multifocal variant of a particular lens material, respectively. Abbreviation: PBS, phosphate-buffered saline.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: In vitro release of two anti-muscarinic drugs from soft contact lenses

    doi: 10.2147/OPTH.S141404

    Figure Lengend Snippet: Pirenzepine release over 24 hours in 4 mL of PBS from contact lenses after 24 hours of uptake in 1 mg/mL pirenzepine solution. Notes: Bars represent standard deviation. ▲, etafilcon A; ◆, ocufilcon B; □, omafilcon B (MF); ○, omafilcon A (SV); Δ, omafilcon A (MF); ●, narafilcon A; ✹, nelfilcon A (SV); ◇, nelfilcon A (MF); ⋆, comfilcon A (MF); ∇, delefilcon A (n=4/ material). SV and MF denote the single vision and multifocal variant of a particular lens material, respectively. Abbreviation: PBS, phosphate-buffered saline.

    Article Snippet: Reagents and materials Atropine sulphate, pirenzepine dihydrochloride and 10× phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Co. (Oakville, ON, Canada).

    Techniques: Standard Deviation, Variant Assay

    Pirenzepine release over 24 hours in 4 mL of PBS from contact lenses after 24 hours of uptake in 10 mg/mL pirenzepine solution. Notes: Bars represent standard deviation. ▲, etafilcon A; ◆, ocufilcon B; □, omafilcon B (MF); ○, omafilcon A (SV); Δ, omafilcon A (MF); ●, narafilcon A; ✹, nelfilcon A (SV); ◇, nelfilcon A (MF); ⋆, comfilcon A (MF); ∇, delefilcon A (n=4/ material). SV and MF denote the single vision and multifocal variant of a particular lens material, respectively. Abbreviation: PBS, phosphate-buffered saline.

    Journal: Clinical Ophthalmology (Auckland, N.Z.)

    Article Title: In vitro release of two anti-muscarinic drugs from soft contact lenses

    doi: 10.2147/OPTH.S141404

    Figure Lengend Snippet: Pirenzepine release over 24 hours in 4 mL of PBS from contact lenses after 24 hours of uptake in 10 mg/mL pirenzepine solution. Notes: Bars represent standard deviation. ▲, etafilcon A; ◆, ocufilcon B; □, omafilcon B (MF); ○, omafilcon A (SV); Δ, omafilcon A (MF); ●, narafilcon A; ✹, nelfilcon A (SV); ◇, nelfilcon A (MF); ⋆, comfilcon A (MF); ∇, delefilcon A (n=4/ material). SV and MF denote the single vision and multifocal variant of a particular lens material, respectively. Abbreviation: PBS, phosphate-buffered saline.

    Article Snippet: Reagents and materials Atropine sulphate, pirenzepine dihydrochloride and 10× phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Co. (Oakville, ON, Canada).

    Techniques: Standard Deviation, Variant Assay

    Dose–response curves for the overt nociception caused by i.pl. injection of BK, Tyr 8 -BK, des-Arg 9 -BK, resiniferatoxin, or capsaicin in mice. The effects of the drugs are expressed as licking time (s). Each point on the curve represents the mean of 4–6 animals and vertical lines show the s.e.m. Asterisks denote the significance levels in comparison with control groups (PBS for kinins or vehicle for vanilloids; one-way ANOVA followed by Dunnett's test): * P

    Journal: British Journal of Pharmacology

    Article Title: Contribution of vanilloid receptors to the overt nociception induced by B2 kinin receptor activation in mice

    doi: 10.1038/sj.bjp.0705546

    Figure Lengend Snippet: Dose–response curves for the overt nociception caused by i.pl. injection of BK, Tyr 8 -BK, des-Arg 9 -BK, resiniferatoxin, or capsaicin in mice. The effects of the drugs are expressed as licking time (s). Each point on the curve represents the mean of 4–6 animals and vertical lines show the s.e.m. Asterisks denote the significance levels in comparison with control groups (PBS for kinins or vehicle for vanilloids; one-way ANOVA followed by Dunnett's test): * P

    Article Snippet: The following drugs were used: BK, Tyr8 -BK, des-Arg9 -BK, des-Arg9 -Leu8 -BK, resiniferatoxin, baicalein, lactic acid, PBS tablets (all from Sigma Chemical Company, St Louis, MO, U.S.A.), GF109203X, , PACOCF3 (Tocris, Ellisville, MO, U.S.A.), capsaicin and capsazepine (RBI, Natick, MA, U.S.A.).

    Techniques: Injection, Mouse Assay

    (a) C 1s spectrum of an as-prepared OEG-NPEOC-APTES film. (b) C 1s spectrum after photodeprotection by exposure to UV light at 244 nm and immersion in PBS solution. (c,d) C 1s spectra of, respectively, an APTES film and a deprotected OEG-NPEOC-APTES film following derivatization by reaction with TFAA. (e) C 1s spectrum of a deprotected OEG-NPEOC-APTES film after incubation with glutaraldehyde solution. (f) C 1s spectrum acquired after subsequent reaction of the aldehyde-functionalized surface with ABNTA. (g) Ni 2p spectrum after incubation of an NTA-functionalized surface similar to that in panel f with nickel chloride solution.

    Journal: Langmuir

    Article Title: Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups

    doi: 10.1021/acs.langmuir.5b04368

    Figure Lengend Snippet: (a) C 1s spectrum of an as-prepared OEG-NPEOC-APTES film. (b) C 1s spectrum after photodeprotection by exposure to UV light at 244 nm and immersion in PBS solution. (c,d) C 1s spectra of, respectively, an APTES film and a deprotected OEG-NPEOC-APTES film following derivatization by reaction with TFAA. (e) C 1s spectrum of a deprotected OEG-NPEOC-APTES film after incubation with glutaraldehyde solution. (f) C 1s spectrum acquired after subsequent reaction of the aldehyde-functionalized surface with ABNTA. (g) Ni 2p spectrum after incubation of an NTA-functionalized surface similar to that in panel f with nickel chloride solution.

    Article Snippet: HS(CH2 )11 (EG)3 NTA was purchased from Prochimia Surfaces (Sopot, Poland). (3-Aminopropyl)triethoxysilane (APTES, 99%) and phosphate-buffered saline (PBS) tablets were supplied by Sigma-Aldrich (Poole, U.K.).

    Techniques: Incubation

    GABA probe calibration in different concentrations of GABA (5, 10, 20, and 40 μM) and α-ketoglutarate (5, 10, 20, 40, 100, 200, and 500 μM) in 1X PBS. (A) Current response at GABA microbiosensor in Site 2 and Glu microbiosensor in Site 1 in PBS only (background or control – red dashed curve, blue dashed curve, respectively) and in 100 μM α-ketoglutarate in 1X PBS (red and blue solid curves, respectively). (B) Current response at GABA microbiosensor for other concentrations of α-ketoglutarate. (C,D) Current response and linear fitting at GABA microbiosensor for different GABA concentrations and α-ketoglutarate concentrations at ≥ 40 μM. Legends: 40 μM (red), 100 μM (green), 200 μM (blue) and 500 μM (magenta). The microbiosensors were biased at + 0.7 V vs Ag/AgCl reference. The solution was stirred at 200 rpm and maintained at 37°C. Linear fit parameters obtained: 40 μM α-keto sensitivity = (0.55 ± 0.077 pA/μM), R 2 = 0.99728; 100 μM α-keto sensitivity = (1.74 ± 0.13 pA/μM), R 2 = 0.99582; 200 μM α-keto sensitivity = (1.03 ± 0.13 pA/μM), R 2 = 0.99582 and 500 μM α-keto sensitivity = (1.51 ± 0.13 pA/μM); R 2 = 0.99582 at GABA microbiosensor. Two-tailed Students t -test was performed ( n = 6, p

    Journal: Frontiers in Neuroscience

    Article Title: A Novel Microbiosensor Microarray for Continuous ex Vivo Monitoring of Gamma-Aminobutyric Acid in Real-Time

    doi: 10.3389/fnins.2018.00500

    Figure Lengend Snippet: GABA probe calibration in different concentrations of GABA (5, 10, 20, and 40 μM) and α-ketoglutarate (5, 10, 20, 40, 100, 200, and 500 μM) in 1X PBS. (A) Current response at GABA microbiosensor in Site 2 and Glu microbiosensor in Site 1 in PBS only (background or control – red dashed curve, blue dashed curve, respectively) and in 100 μM α-ketoglutarate in 1X PBS (red and blue solid curves, respectively). (B) Current response at GABA microbiosensor for other concentrations of α-ketoglutarate. (C,D) Current response and linear fitting at GABA microbiosensor for different GABA concentrations and α-ketoglutarate concentrations at ≥ 40 μM. Legends: 40 μM (red), 100 μM (green), 200 μM (blue) and 500 μM (magenta). The microbiosensors were biased at + 0.7 V vs Ag/AgCl reference. The solution was stirred at 200 rpm and maintained at 37°C. Linear fit parameters obtained: 40 μM α-keto sensitivity = (0.55 ± 0.077 pA/μM), R 2 = 0.99728; 100 μM α-keto sensitivity = (1.74 ± 0.13 pA/μM), R 2 = 0.99582; 200 μM α-keto sensitivity = (1.03 ± 0.13 pA/μM), R 2 = 0.99582 and 500 μM α-keto sensitivity = (1.51 ± 0.13 pA/μM); R 2 = 0.99582 at GABA microbiosensor. Two-tailed Students t -test was performed ( n = 6, p

    Article Snippet: Chemicals Phosphate buffered saline (PBS), bovine serum albumin (BSA), glutaraldehyde, GABA, GABASE from Pseudomonas fluorescens and α-ketoglutarate disodium salt was purchased from Millipore-Sigma (MO, United States).

    Techniques: Two Tailed Test

    Comparison of mRNA expression induced by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in MLE-15 cells. MLE-15 cells were treated with 0–100 µ g/m l LPS (A) or LTA (B) at 37°C for 2 hr. MLE-15 cells treated with phosphate-buffered saline (PBS) served as a control. mRNA expression of SAA1/2 and SAA3 was normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA expression and compared with that in control cells assumed to have 0 µ g/m l expression. Data are the mean plus standard deviation from three independent experiments. ** P

    Journal: The Journal of Veterinary Medical Science

    Article Title: Lipopolysaccharide and lipoteichoic acid enhance serum amyloid A3 mRNA expression in murine alveolar epithelial cells

    doi: 10.1292/jvms.19-0154

    Figure Lengend Snippet: Comparison of mRNA expression induced by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) in MLE-15 cells. MLE-15 cells were treated with 0–100 µ g/m l LPS (A) or LTA (B) at 37°C for 2 hr. MLE-15 cells treated with phosphate-buffered saline (PBS) served as a control. mRNA expression of SAA1/2 and SAA3 was normalized to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) mRNA expression and compared with that in control cells assumed to have 0 µ g/m l expression. Data are the mean plus standard deviation from three independent experiments. ** P

    Article Snippet: After incubation, the cells were rinsed with sterile phosphate-buffered saline (PBS) and treated with LPS from E. coli O111:B4 (115K4092, Sigma, St. Louis, MO, U.S.A.) or lipoteichoic acid (LTA) from Bacillus subtilis (L3265-5MG, Sigma), which are gram-negative and -positive bacterial membranous antigens, respectively.

    Techniques: Expressing, Standard Deviation

    Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.

    Journal: PLoS ONE

    Article Title: CXCR4 Is Dispensable for T Cell Egress from Chronically Inflamed Skin via the Afferent Lymph

    doi: 10.1371/journal.pone.0095626

    Figure Lengend Snippet: Pharmacological inhibition of CXCR4 does not impact the tissue exit of wildtype or Ccr7 −/− T cells. ( A–B ) Chemotaxis of mouse splenocytes was tested toward mouse CXCL12 ( A ) and CCL21 ( B ) in a Transwell chemotaxis assay in the presence or absence of CXCR4 inhibitor AMD3100 at indicated concentrations. Data represent mean ±SD of triplicate wells. One of two similar experiments is shown. ( C–E ) Mice carrying 3-week-old skin granulomas in the footpads were systemically treated with either PBS or 1000 µg/kg/h AMD3100 through subcutaneously implanted mini osmotic pumps. 12 h after implantation, a mixture of fluorescently labeled Ccr7 −/− and wildtype splenocytes were transferred into the inflamed footpads. The numbers and phenotypes of cells that egressed from the skin and entered the draining lymph node were determined by flow cytometry 12 h after transfer. The numbers of wildtype ( C ) and Ccr7 −/− ( D ) lymphocyte subsets that migrated to the draining lymph nodes are shown. Data points represent individually analyzed mice of groups of 7–10 recipient mice per group; horizontal lines indicate the mean of each group. One of two experiments with similar results ( C and D ) or both experiments combined ( E ) are shown. WT, wildtype.

    Article Snippet: Sterile-filtered AMD3100 in PBS (Sigma-Aldrich) or PBS alone (Invitrogen) was loaded into the osmotic pumps before equilibration in sterile PBS at 37°C for 12 h according to the manufacturer’s instructions.

    Techniques: Inhibition, Chemotaxis Assay, Mouse Assay, Labeling, Flow Cytometry, Cytometry

    EMT with proliferation induced by EGTA with EGF+FGF-2 is caused by Wnt/β-catenin signaling by rescued by SS3Y β-catenin. (a) The TCF/LEF reporter activity was silent in cells treated by PBS, EGTA, EGF, FGF-2, TGF-β1, or EGF+FGF-2+TGF-β1 but elevated 15-fold by EGTA with EGF+FGF-2. The latter was abolished by XAV939 (*, P

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Wnt Signaling Induces Epithelial-Mesenchymal Transition with Proliferation in ARPE-19 Cells upon Loss of Contact Inhibition

    doi: 10.1038/labinvest.2011.201

    Figure Lengend Snippet: EMT with proliferation induced by EGTA with EGF+FGF-2 is caused by Wnt/β-catenin signaling by rescued by SS3Y β-catenin. (a) The TCF/LEF reporter activity was silent in cells treated by PBS, EGTA, EGF, FGF-2, TGF-β1, or EGF+FGF-2+TGF-β1 but elevated 15-fold by EGTA with EGF+FGF-2. The latter was abolished by XAV939 (*, P

    Article Snippet: Antibodies and Reagents Dulbecco’s modified Eagle’s medium (DMEM), Ham’s/F12 medium, human epidermal growth factor (EGF), HEPES buffer, phosphate-buffered saline (PBS), amphotericin B, gentamicin, fetal bovine serum (FBS), and Alexa fluor-conjugated secondary IgG were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Activity Assay

    Contact inhibition is unlocked by EGTA only in the presence of EGF and/or FGF-2, but not TGF-β1. (a) Immunostaining of BrdU was performed in ARPE-19 cells cultured to day 7 post-confluence after being treated without or with 1mg/mL EGTA immediately followed by PBS, 10 ng/mL EGF, 20 ng/mL FGF-2, 10 ng/mL TGF-β1, 10 ng/mL EGF + 20 ng/mL FGF-2, or 10 ng/mL EGF + 20 ng/mL FGF-2 + 10 ng/mL TGF-β1 for 1 day. Without EGTA, no BrdU labeling was found. When EGTA was added, BrdU labeling was promoted by EGF, FGF-2, or EGF+FGF-2, but not by PBS, TGF-β1 or EGF+FGF-2+TGF-β1. Scale bar, 100 μm. (b) The BrdU labeling index was significantly increased by EGF, FGF-2, or in combination (highest) when EGTA was added (*, †, and ‡, P

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Wnt Signaling Induces Epithelial-Mesenchymal Transition with Proliferation in ARPE-19 Cells upon Loss of Contact Inhibition

    doi: 10.1038/labinvest.2011.201

    Figure Lengend Snippet: Contact inhibition is unlocked by EGTA only in the presence of EGF and/or FGF-2, but not TGF-β1. (a) Immunostaining of BrdU was performed in ARPE-19 cells cultured to day 7 post-confluence after being treated without or with 1mg/mL EGTA immediately followed by PBS, 10 ng/mL EGF, 20 ng/mL FGF-2, 10 ng/mL TGF-β1, 10 ng/mL EGF + 20 ng/mL FGF-2, or 10 ng/mL EGF + 20 ng/mL FGF-2 + 10 ng/mL TGF-β1 for 1 day. Without EGTA, no BrdU labeling was found. When EGTA was added, BrdU labeling was promoted by EGF, FGF-2, or EGF+FGF-2, but not by PBS, TGF-β1 or EGF+FGF-2+TGF-β1. Scale bar, 100 μm. (b) The BrdU labeling index was significantly increased by EGF, FGF-2, or in combination (highest) when EGTA was added (*, †, and ‡, P

    Article Snippet: Antibodies and Reagents Dulbecco’s modified Eagle’s medium (DMEM), Ham’s/F12 medium, human epidermal growth factor (EGF), HEPES buffer, phosphate-buffered saline (PBS), amphotericin B, gentamicin, fetal bovine serum (FBS), and Alexa fluor-conjugated secondary IgG were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Inhibition, Immunostaining, Cell Culture, Labeling

    EGTA plus EGF+FGF-2 activates Wnt/β-catenin signaling. (a) Immunostaining revealed nuclear β-catenin and LEF1 only when cells were treated with EGTA plus EGF+FGF-2 but not EGTA alone. Scale bar, 100 μm (b) Western blot analysis confirmed increased nuclear contents of β-catenin and LEF1 in the nuclear extract, but decreased β-catenin in the membranous extract when cells were treated by EGTA with EGF+FGF-2. Levels of connexin 43, α-tubulin, and histone were used as loading controls for membranous, cytosolic and nuclear extracts, respectively. (c) Overexpression of S33Y β-catenin in post-confluent ARPE treated with EGTA without growth factors increased BrdU labeling, nuclear β-catenin and S100A4, and cytoplasmic α-SMA. (d) Immunoprecipitation by anti-β-catenin antibody followed by Western blotting with anti-LEF-1 antibody confirmed tight association between β-catenin and LEF-1 in the nuclear extract from EGTA plus EGF+FGF-2 but not from PBS or EGTA alone.

    Journal: Laboratory investigation; a journal of technical methods and pathology

    Article Title: Wnt Signaling Induces Epithelial-Mesenchymal Transition with Proliferation in ARPE-19 Cells upon Loss of Contact Inhibition

    doi: 10.1038/labinvest.2011.201

    Figure Lengend Snippet: EGTA plus EGF+FGF-2 activates Wnt/β-catenin signaling. (a) Immunostaining revealed nuclear β-catenin and LEF1 only when cells were treated with EGTA plus EGF+FGF-2 but not EGTA alone. Scale bar, 100 μm (b) Western blot analysis confirmed increased nuclear contents of β-catenin and LEF1 in the nuclear extract, but decreased β-catenin in the membranous extract when cells were treated by EGTA with EGF+FGF-2. Levels of connexin 43, α-tubulin, and histone were used as loading controls for membranous, cytosolic and nuclear extracts, respectively. (c) Overexpression of S33Y β-catenin in post-confluent ARPE treated with EGTA without growth factors increased BrdU labeling, nuclear β-catenin and S100A4, and cytoplasmic α-SMA. (d) Immunoprecipitation by anti-β-catenin antibody followed by Western blotting with anti-LEF-1 antibody confirmed tight association between β-catenin and LEF-1 in the nuclear extract from EGTA plus EGF+FGF-2 but not from PBS or EGTA alone.

    Article Snippet: Antibodies and Reagents Dulbecco’s modified Eagle’s medium (DMEM), Ham’s/F12 medium, human epidermal growth factor (EGF), HEPES buffer, phosphate-buffered saline (PBS), amphotericin B, gentamicin, fetal bovine serum (FBS), and Alexa fluor-conjugated secondary IgG were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Immunostaining, Western Blot, Over Expression, Labeling, Immunoprecipitation

    Calorimetry data for the titration of OD4.2.2 with VRC-PG04 antibody in PBS buffer (pH 7.4). Measurements were carried out in triplicate with a representative titration result shown. The top panel shows raw data with the area under each spike proportional to the heat produced at each injection of VRC-PG04 Fab. The lower panel shows integrated areas normalized to the number of moles of VRC-PG04 Fab.

    Journal: Journal of Virology

    Article Title: Outer Domain of HIV-1 gp120: Antigenic Optimization, Structural Malleability, and Crystal Structure with Antibody VRC-PG04

    doi: 10.1128/JVI.02717-12

    Figure Lengend Snippet: Calorimetry data for the titration of OD4.2.2 with VRC-PG04 antibody in PBS buffer (pH 7.4). Measurements were carried out in triplicate with a representative titration result shown. The top panel shows raw data with the area under each spike proportional to the heat produced at each injection of VRC-PG04 Fab. The lower panel shows integrated areas normalized to the number of moles of VRC-PG04 Fab.

    Article Snippet: OD4.2.2 and VRC-PG04 Fab were dialyzed in the same PBS buffer (Gibco) overnight at room temperature.

    Techniques: Titration, Produced, Injection

    (A) 34DABA-coated SPIOs are dispersed in EG, DEG, TEG, GC, PBS, DMEM, DMEM + 5% FBS, and FBS with corresponding markings of NBFF-1, NBFF-2, NBFF-3, NBFF-4, BFF-1, BFF-2, BFF-3, and BFF-4. (B) SAR values of NBFF-1, NBFF-2, NBFF-3, NBFF-4, BFF-1, BFF-2, BFF-3, and BFF-4 at 0.5 mg mL –1 concentration on exposure to AMF with H × f value of 8.2 GAm –1 s –1 .

    Journal: ACS Omega

    Article Title: Functionalized Hydrophilic Superparamagnetic Iron Oxide Nanoparticles for Magnetic Fluid Hyperthermia Application in Liver Cancer Treatment

    doi: 10.1021/acsomega.8b00207

    Figure Lengend Snippet: (A) 34DABA-coated SPIOs are dispersed in EG, DEG, TEG, GC, PBS, DMEM, DMEM + 5% FBS, and FBS with corresponding markings of NBFF-1, NBFF-2, NBFF-3, NBFF-4, BFF-1, BFF-2, BFF-3, and BFF-4. (B) SAR values of NBFF-1, NBFF-2, NBFF-3, NBFF-4, BFF-1, BFF-2, BFF-3, and BFF-4 at 0.5 mg mL –1 concentration on exposure to AMF with H × f value of 8.2 GAm –1 s –1 .

    Article Snippet: Phosphate buffer saline (PBS), fetal bovine serum (FBS), and Dulbecco’s modified eagle medium (DMEM) are purchased from Gibco Life technologies.

    Techniques: Concentration Assay

    Imject- and Alhydrogel Alum-adsorbed CTD1 stimulates long-term Bmem responses. Mice were not immunized (naive) or were immunized with 50 μg CTD1 in PBS, CTD1 adsorbed to Imject Alum, or CTD1 adsorbed to Alhydrogel. Mice were rested for 180 days

    Journal: Infection and Immunity

    Article Title: Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B

    doi: 10.1128/IAI.00011-15

    Figure Lengend Snippet: Imject- and Alhydrogel Alum-adsorbed CTD1 stimulates long-term Bmem responses. Mice were not immunized (naive) or were immunized with 50 μg CTD1 in PBS, CTD1 adsorbed to Imject Alum, or CTD1 adsorbed to Alhydrogel. Mice were rested for 180 days

    Article Snippet: Vaccines consisted of 25, 50, or 100 μg of CTD in 200 μl sterile phosphate-buffered saline (PBS) adsorbed to Imject Alum (Thermo Scientific, Rockford, IL) or Alhydrogel Alum (Invivogen, San Diego, CA).

    Techniques: Mouse Assay

    Imject Alum-adsorbed CTD1 stimulates primary and recall IgG1 responses. C57BL/6 mice were immunized s.c. with Imject-adsorbed CTD1. Mice received a booster vaccine consisting of CTD1 in PBS on day 60. Sera were collected at the times indicated, and endpoint

    Journal: Infection and Immunity

    Article Title: Memory B Cells Encode Neutralizing Antibody Specific for Toxin B from the Clostridium difficile Strains VPI 10463 and NAP1/BI/027 but with Superior Neutralization of VPI 10463 Toxin B

    doi: 10.1128/IAI.00011-15

    Figure Lengend Snippet: Imject Alum-adsorbed CTD1 stimulates primary and recall IgG1 responses. C57BL/6 mice were immunized s.c. with Imject-adsorbed CTD1. Mice received a booster vaccine consisting of CTD1 in PBS on day 60. Sera were collected at the times indicated, and endpoint

    Article Snippet: Vaccines consisted of 25, 50, or 100 μg of CTD in 200 μl sterile phosphate-buffered saline (PBS) adsorbed to Imject Alum (Thermo Scientific, Rockford, IL) or Alhydrogel Alum (Invivogen, San Diego, CA).

    Techniques: Mouse Assay

    CAP256 gp140-FL-IP-His protein α-Env ELISA. Binding ELISAs using anti-Env human monoclonal antibodies to CAP256 gp140-FL-IP-His. (A) PGT128 and PGT135 binding indicate the presence of the Env V3-glycan supersite. Similarly, the CD4 binding site using VRC01 The V2-glycan epitope was detected with PG9 (A), but no binding was observed for the native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08 (B). In line with this, clear ELISA signals were observed for F105 and 446-52D, indicative of the presence of misfolded Env (A). (C) Control soluble, trimeric BG505_664-His Env protein performed as expected, with ELISA signals for native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08. (D) No binding of MAbs was observed for the no protein (PBS) control.

    Journal: PLoS ONE

    Article Title: The adjuvant AlhydroGel elicits higher antibody titres than AddaVax when combined with HIV-1 subtype C gp140 from CAP256

    doi: 10.1371/journal.pone.0208310

    Figure Lengend Snippet: CAP256 gp140-FL-IP-His protein α-Env ELISA. Binding ELISAs using anti-Env human monoclonal antibodies to CAP256 gp140-FL-IP-His. (A) PGT128 and PGT135 binding indicate the presence of the Env V3-glycan supersite. Similarly, the CD4 binding site using VRC01 The V2-glycan epitope was detected with PG9 (A), but no binding was observed for the native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08 (B). In line with this, clear ELISA signals were observed for F105 and 446-52D, indicative of the presence of misfolded Env (A). (C) Control soluble, trimeric BG505_664-His Env protein performed as expected, with ELISA signals for native-like trimer-specific MAbs PG16, PGT145 and (CAP256) VRC26_08. (D) No binding of MAbs was observed for the no protein (PBS) control.

    Article Snippet: ELISA plates were washed with PBS (Lonza, Basel) and blocked using 5% non-fat milk (Sigma, St Louis) in PBS.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Cell - Type Specific Requirements for Thioredoxin and / or PDI in HIV - 1 Infection in Primary Human Cells. PHA stimulated PBL were pre-incubated for 1 h with relevant mAbs (50 μg/ml) and then infected for 2.5 h with HIV-1 JR-FL Env pseudotyped, Luc reporter gene encoding virus particles in the continued presence of the mAbs. After infection, the cells were washed and further incubated for 48 h in RPMI supplemented with 10% FCS and L-Glutamine prior to lysis (0.5% Triton-X100 in PBS) and Luc activity was then measured. MDM were treated and infected in an identical manner, using DMEM medium. The amount of HIV-1 Env pseudotyped virus particles was adjusted according to the number of cells subjected to infection. Primary human MDM or PBLs from four different donors were prepared and tested as described in Methods. The RLU established for the anti-PDI or anti-Trx mAbs treated cells were calculated as a percent of the mean Luc activity of cells pre-incubated and infected in the presence of nonspecific mouse IgG. The means of the four established values from each donor for the anti-PDI or anti-Trx mAbs treated PBL and MDM are presented in the left and right panel of Figure 5 , respectively.

    Journal: Retrovirology

    Article Title: Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    doi: 10.1186/1742-4690-9-97

    Figure Lengend Snippet: Cell - Type Specific Requirements for Thioredoxin and / or PDI in HIV - 1 Infection in Primary Human Cells. PHA stimulated PBL were pre-incubated for 1 h with relevant mAbs (50 μg/ml) and then infected for 2.5 h with HIV-1 JR-FL Env pseudotyped, Luc reporter gene encoding virus particles in the continued presence of the mAbs. After infection, the cells were washed and further incubated for 48 h in RPMI supplemented with 10% FCS and L-Glutamine prior to lysis (0.5% Triton-X100 in PBS) and Luc activity was then measured. MDM were treated and infected in an identical manner, using DMEM medium. The amount of HIV-1 Env pseudotyped virus particles was adjusted according to the number of cells subjected to infection. Primary human MDM or PBLs from four different donors were prepared and tested as described in Methods. The RLU established for the anti-PDI or anti-Trx mAbs treated cells were calculated as a percent of the mean Luc activity of cells pre-incubated and infected in the presence of nonspecific mouse IgG. The means of the four established values from each donor for the anti-PDI or anti-Trx mAbs treated PBL and MDM are presented in the left and right panel of Figure 5 , respectively.

    Article Snippet: Phosphate Buffered Saline (PBS) and RPMI 1640 medium were purchased from Lonza (Walkersville, MD).

    Techniques: Infection, Incubation, Lysis, Activity Assay

    Anti - PDI, but not anti - Thioredoxin mAbs inhibit HIV - 1 infection in PM - 1 T - cell line. A . PM-1 cells were pre-incubated for 1 h in RPMI-1640 containing the indicated concentrations of nonspecific mouse IgG, anti-PDI or anti-Trx mAbs prior to infection with HIV-1 JR-FL Env pseudotyped, Luc reporter gene-encoding virus particles for 2.5 h in the presence of the different mAbs. After the infection period, cells were washed and further incubated for 48 h in RC-10 before being lysed with 0.5% Triton-X100 in PBS and Luc activity was measured. B , C . Following preincubation for 30 minutes with 5 mM DTNB, anti-PDI or anti-Trx mAbs, PM-1cells were infected with HIV-1 ADA (panel B ) or HIV-1 LAV (panel C ). After infection for 2 h in the presence of the aforementioned inhibitors, the PM-1 cells were washed and further incubated in RC-10 without DTNB or mAbs. Cell culture supernatants were collected every two days and cryopreserved before being evaluated for RT activity.

    Journal: Retrovirology

    Article Title: Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    doi: 10.1186/1742-4690-9-97

    Figure Lengend Snippet: Anti - PDI, but not anti - Thioredoxin mAbs inhibit HIV - 1 infection in PM - 1 T - cell line. A . PM-1 cells were pre-incubated for 1 h in RPMI-1640 containing the indicated concentrations of nonspecific mouse IgG, anti-PDI or anti-Trx mAbs prior to infection with HIV-1 JR-FL Env pseudotyped, Luc reporter gene-encoding virus particles for 2.5 h in the presence of the different mAbs. After the infection period, cells were washed and further incubated for 48 h in RC-10 before being lysed with 0.5% Triton-X100 in PBS and Luc activity was measured. B , C . Following preincubation for 30 minutes with 5 mM DTNB, anti-PDI or anti-Trx mAbs, PM-1cells were infected with HIV-1 ADA (panel B ) or HIV-1 LAV (panel C ). After infection for 2 h in the presence of the aforementioned inhibitors, the PM-1 cells were washed and further incubated in RC-10 without DTNB or mAbs. Cell culture supernatants were collected every two days and cryopreserved before being evaluated for RT activity.

    Article Snippet: Phosphate Buffered Saline (PBS) and RPMI 1640 medium were purchased from Lonza (Walkersville, MD).

    Techniques: Infection, Incubation, Activity Assay, Cell Culture

    Comparison of inflammatory parameters for cobalt monoxide (CoO) nanoparticles (NPs) and cobalt chloride (CoCl 2 ). Number of ( A ) total cells, ( B ) macrophages, ( C ) neutrophils, ( D ) eosinophils, and levels of ( E ) LDH, ( F ) total protein, ( G ) IL-6, and ( H ) eotaxin. Both treatment groups had same doses for cobalt ions (31, 79, and 315 μg/rat). Note that the inflammatory parameters of CoO NPs were similar with the CoCl 2 which implies that the toxicity of CoO NPs was derived from their solubilized cobalt ions. Values are mean ± SEM ( n = 4) for each treatment group. The data from CoO NPs were compared with the vehicle control (Ca 2+ - and Mg 2+ -free phosphate buffered saline) to determine statistical significance. * p

    Journal: Particle and Fibre Toxicology

    Article Title: Response-metrics for acute lung inflammation pattern by cobalt-based nanoparticles

    doi: 10.1186/s12989-015-0089-1

    Figure Lengend Snippet: Comparison of inflammatory parameters for cobalt monoxide (CoO) nanoparticles (NPs) and cobalt chloride (CoCl 2 ). Number of ( A ) total cells, ( B ) macrophages, ( C ) neutrophils, ( D ) eosinophils, and levels of ( E ) LDH, ( F ) total protein, ( G ) IL-6, and ( H ) eotaxin. Both treatment groups had same doses for cobalt ions (31, 79, and 315 μg/rat). Note that the inflammatory parameters of CoO NPs were similar with the CoCl 2 which implies that the toxicity of CoO NPs was derived from their solubilized cobalt ions. Values are mean ± SEM ( n = 4) for each treatment group. The data from CoO NPs were compared with the vehicle control (Ca 2+ - and Mg 2+ -free phosphate buffered saline) to determine statistical significance. * p

    Article Snippet: NP suspensions at 80, 200, and 800 μg/mL were prepared by dispersing NPs in sterile Ca2+ - and Mg2+ -free PBS (Life Technologies, Gaithersburg, MD, USA) and sonicated for 10 min using a bath sonicator (Saehan-Sonic, Seoul, Korea) to break up agglomerates.

    Techniques: Derivative Assay

    Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to PBS and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to PBS and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: Labeling, Mouse Assay, Software

    Multipotent adult progenitor cells (MAPC) suppress IL-7-induced interferon-γ (IFN-γ) production by T cells in vivo . Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (A) The frequency of IFN-γ producing CD4 + and CD8 + in the spleen was significantly increased following IL-7 administration. Both MAPC IP and MAPC IV reduced IFN-γ production by T cells (PBS: n = 8, PBS + MAPC IP: n = 12, PBS + MAPC IV: n = 8, IL-7: n = 6, IL-7 + MAPC IP: n = 11, IL-7 + MAPC IV: n = 8). (B) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of IFN-γ production by CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 8, PBS + MAPC IP: n = 11, PBS + MAPC IV: n = 7, IL-7: n = 7, IL-7 + MAPC IP: n = 10, and IL-7 + MAPC IV: n = 7). Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) suppress IL-7-induced interferon-γ (IFN-γ) production by T cells in vivo . Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (A) The frequency of IFN-γ producing CD4 + and CD8 + in the spleen was significantly increased following IL-7 administration. Both MAPC IP and MAPC IV reduced IFN-γ production by T cells (PBS: n = 8, PBS + MAPC IP: n = 12, PBS + MAPC IV: n = 8, IL-7: n = 6, IL-7 + MAPC IP: n = 11, IL-7 + MAPC IV: n = 8). (B) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of IFN-γ production by CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 8, PBS + MAPC IP: n = 11, PBS + MAPC IV: n = 7, IL-7: n = 7, IL-7 + MAPC IP: n = 10, and IL-7 + MAPC IV: n = 7). Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: In Vivo, Recombinant

    Multipotent adult progenitor cells (MAPC) have no effect on T cell proliferation following lymphodepletion. 50 mg/kg anti-thymocyte globulin (ATG) was administered intraperitoneally (IP) on days 0 and 3. (A) Spleens and lymph nodes were harvested on days 4 and 7, and total numbers of CD4 + and CD8 + cells were quantified using counting beads and flow cytometry. (B) Schematic timeline of experiments with MAPC (1 × 10 6 ) administration [IP or intravenously (IV)] to the ATG model on day 4. (C) MAPC administered IV or IP had no effect on the proliferation of either CD4 + or CD8 + T cells in the spleens and lymph nodes following ATG administration (spleens; PBS: n = 10, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 8 and lymph nodes; PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 9, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. *

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) have no effect on T cell proliferation following lymphodepletion. 50 mg/kg anti-thymocyte globulin (ATG) was administered intraperitoneally (IP) on days 0 and 3. (A) Spleens and lymph nodes were harvested on days 4 and 7, and total numbers of CD4 + and CD8 + cells were quantified using counting beads and flow cytometry. (B) Schematic timeline of experiments with MAPC (1 × 10 6 ) administration [IP or intravenously (IV)] to the ATG model on day 4. (C) MAPC administered IV or IP had no effect on the proliferation of either CD4 + or CD8 + T cells in the spleens and lymph nodes following ATG administration (spleens; PBS: n = 10, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 8 and lymph nodes; PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 9, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. *

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry

    Multipotent adult progenitor cells (MAPC) promote Treg frequency and suppress interferon-γ (IFN-γ) production by T cells following lymphodepletion. (A) Bar graph and representative FACS plots demonstrate that anti-thymocyte globulin (ATG) increased the frequency of CD4 + , CD25 + , and FoxP3 + T cells in the spleen and lymph nodes, and MAPC cells administered intraperitoneally (MAPC IP) further increased this in the lymph nodes [PBS: n = 13, ATG: n = 13, ATG + MAPC IP: n = 11, and ATG + MAPC cells administered intravenously (MAPC IV): n = 13]. (B) MAPC IP reduced the frequency of IFN-γ producing CD4 + and CD8 + T cells in the spleen (PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 10). (C) MAPC IP reduced the frequency of IFN-γ producing CD8 + T cells but not CD4 + T cells in the lymph nodes (PBS: n = 12, ATG: n = 10, ATG + MAPC IP: n = 11, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) promote Treg frequency and suppress interferon-γ (IFN-γ) production by T cells following lymphodepletion. (A) Bar graph and representative FACS plots demonstrate that anti-thymocyte globulin (ATG) increased the frequency of CD4 + , CD25 + , and FoxP3 + T cells in the spleen and lymph nodes, and MAPC cells administered intraperitoneally (MAPC IP) further increased this in the lymph nodes [PBS: n = 13, ATG: n = 13, ATG + MAPC IP: n = 11, and ATG + MAPC cells administered intravenously (MAPC IV): n = 13]. (B) MAPC IP reduced the frequency of IFN-γ producing CD4 + and CD8 + T cells in the spleen (PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 10). (C) MAPC IP reduced the frequency of IFN-γ producing CD8 + T cells but not CD4 + T cells in the lymph nodes (PBS: n = 12, ATG: n = 10, ATG + MAPC IP: n = 11, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: FACS

    Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) require prostaglandin E2 (PGE2) to suppress interferon-γ (IFN-γ) production by T cells. 100 mg/kg anti-thymocyte globulin (ATG) or control serum was administered over two doses given on days 0 and 3, followed by the administration of MAPC IP on day 4, and indomethacin (Indo) on days 4, 5, and 6. Spleens and lymph nodes were harvested on day 7 and examined for the production of IFN-γ by T cells. Bar graphs show that suppression of IFN-γ production by CD4 + and CD8 + T cells in the (A) spleen [PBS: n = 7, ATG: n = 7, ATG + MAPC IP: n = 9, and ATG + MAPC intravenous (IV): n = 7] and (B) lymph nodes (PBS: n = 4, ATG: n = 5, ATG + MAPC IP: n = 5, and ATG + MAPC IV: n = 4) was inhibited when Indo was administered on days 4, 5, and 6 of the lymphodepletion model. Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) require prostaglandin E2 (PGE2) to suppress interferon-γ (IFN-γ) production by T cells. 100 mg/kg anti-thymocyte globulin (ATG) or control serum was administered over two doses given on days 0 and 3, followed by the administration of MAPC IP on day 4, and indomethacin (Indo) on days 4, 5, and 6. Spleens and lymph nodes were harvested on day 7 and examined for the production of IFN-γ by T cells. Bar graphs show that suppression of IFN-γ production by CD4 + and CD8 + T cells in the (A) spleen [PBS: n = 7, ATG: n = 7, ATG + MAPC IP: n = 9, and ATG + MAPC intravenous (IV): n = 7] and (B) lymph nodes (PBS: n = 4, ATG: n = 5, ATG + MAPC IP: n = 5, and ATG + MAPC IV: n = 4) was inhibited when Indo was administered on days 4, 5, and 6 of the lymphodepletion model. Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques:

    Multipotent adult progenitor cells (MAPC) suppress IL-7-driven proliferation of T cells in vivo . (A) Schematic timeline of IL-7-driven homeostatic proliferation experiments. Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC cells were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (B) Bar graphs demonstrating that both MAPC IV and MAPC IP reduce the frequency of Ki67 + CD4 + and CD8 + cells in the spleen (PBS: n = 13, PBS + MAPC IP: n = 17, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13) (C) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of Ki67 + CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 12, PBS + MAPC IP: n = 15, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13). Results are indicative of three independent experiments using three MAPC donors * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) suppress IL-7-driven proliferation of T cells in vivo . (A) Schematic timeline of IL-7-driven homeostatic proliferation experiments. Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC cells were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (B) Bar graphs demonstrating that both MAPC IV and MAPC IP reduce the frequency of Ki67 + CD4 + and CD8 + cells in the spleen (PBS: n = 13, PBS + MAPC IP: n = 17, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13) (C) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of Ki67 + CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 12, PBS + MAPC IP: n = 15, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13). Results are indicative of three independent experiments using three MAPC donors * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: In Vivo, Recombinant

    High-sensitivity flow-cytometry analysis of fractionated IDG revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in PBS-loaded (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.

    Journal: Journal of Extracellular Vesicles

    Article Title: A subset of extracellular vesicles carries the bulk of microRNAs in commercial dairy cow’s milk

    doi: 10.1080/20013078.2017.1401897

    Figure Lengend Snippet: High-sensitivity flow-cytometry analysis of fractionated IDG revealed the peculiar enrichment of EVs in the 12,000 g pellet. (a) A pool of all the IDG fractions was labelled with deep red CellTracker fluorescent dye and the gates set for fluorescent EV (green) detection, compared with unlabelled EVs (Red) and CellTracker in PBS-loaded (empty) IDG fractions (PBS, Blue). (b–c) Each IDG fraction of the 12,000 g (b) and 35,000 g (c) was labelled and analysed for the presence of esterase-positive EVs. Results are expressed as a count of events (mean ± SD; n = 3). (d) After setting the gates for size, using beads of known diameter, we analysed the CellTracker positive population in each fraction to determine the approximate size of the observed events. (e–f) Representative results obtained for IDG fractions F7 and F8 from 12,000 g (e) and 35,000 g (f) EVs are displayed. A green colour represents CellTracker positive events. The associated quantification results are available as Supplementary File 5.

    Article Snippet: To this end, 2 µL of each IDG fraction was diluted in 98 µL of 0.2 µm filtered sterile PBS (Gibco, ThermoFisher scientific) containing the fluorescent CellTracker™ deep red dye [ ] (C34565; Life Technologies, ThermoFisher Scientific) at a final concentration of 1 µM, for 20 min at 37°C, in the dark, following the manufacturer’s recommendations.

    Techniques: Flow Cytometry, Cytometry

    The implementation of size-exclusion chromatography to remove Optiprep remnants from EV samples. ( a ) Optiprep density gradients were loaded with PBS to obtain blank EV-density fractions (F1) and with CCM to obtain EV-containing fractions (F2). EVs were retrieved by pelleting at 100,000 g (P1) or by size-exclusion chromatography (S1). Quantity of Optiprep in S1 and P1 was calculated using DC Protein assay. SEC was performed on F2 and consecutive SEC fractions (S2) were analyzed by NTA and protein analysis for GFP and CD9. ( b ) Comparative graph of NTA from S2 (connected line, marked area = SD) and Optiprep quantification of S1 (three differently colored bars representing three independent gradients, SD indicated). ( c ) Protein analysis of S2 by Western blot analysis for GFP and CD9 quantification by TRIFic CD9 assay. Original immunostaining results are shown in Supplementary Fig. 10 . Abbreviations: PBS: phosphate buffered saline. CCM: concentrated conditioned medium. GFP: green fluorescent protein. EV: extracellular vesicle. WB: Western blot. NTA: Nanoparticle tracking analysis. SEC: size-exclusion chromatography.

    Journal: Scientific Reports

    Article Title: Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research

    doi: 10.1038/s41598-017-02599-y

    Figure Lengend Snippet: The implementation of size-exclusion chromatography to remove Optiprep remnants from EV samples. ( a ) Optiprep density gradients were loaded with PBS to obtain blank EV-density fractions (F1) and with CCM to obtain EV-containing fractions (F2). EVs were retrieved by pelleting at 100,000 g (P1) or by size-exclusion chromatography (S1). Quantity of Optiprep in S1 and P1 was calculated using DC Protein assay. SEC was performed on F2 and consecutive SEC fractions (S2) were analyzed by NTA and protein analysis for GFP and CD9. ( b ) Comparative graph of NTA from S2 (connected line, marked area = SD) and Optiprep quantification of S1 (three differently colored bars representing three independent gradients, SD indicated). ( c ) Protein analysis of S2 by Western blot analysis for GFP and CD9 quantification by TRIFic CD9 assay. Original immunostaining results are shown in Supplementary Fig. 10 . Abbreviations: PBS: phosphate buffered saline. CCM: concentrated conditioned medium. GFP: green fluorescent protein. EV: extracellular vesicle. WB: Western blot. NTA: Nanoparticle tracking analysis. SEC: size-exclusion chromatography.

    Article Snippet: A dilution series of 0 to 10% of Optiprep in PBS was obtained as a standard curve for performing absorbance readings with DC Protein assay (Bio-Rad, Hercules, California, USA), according to manufacturer’s instructions.

    Techniques: Size-exclusion Chromatography, DC Protein Assay, Western Blot, Immunostaining

    Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a)  Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS.  (b)  Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA  (c,d)  Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p

    Journal: Biomaterials

    Article Title: Scanningless and continuous 3D bioprinting of human tissues with decellularized extracellular matrix

    doi: 10.1016/j.biomaterials.2018.12.009

    Figure Lengend Snippet: Preparation of photocrosslinkable dECM bioinks and characterization of printed dECM constructs. (a) Lyophilized decellularized tissues were cryomilled and pepsin solubilized followed by a second lyophilization and cryomilling step to yield a fine dECM powder that can be reconstituted with 1X PBS. (b) Schematic of the crosslinking mechanism to form a printed dECM hydrogel construct for mechanically soft heart and liver tissue constructs through the incorporation of GelMA (c,d) Plots of the compressive Young’s modulus as a function of printing exposure time. All data are expressed as mean ± standard deviation. * = significant between exposure times (p

    Article Snippet: Next, tissues were incubated in 4% (w/v) sodium deoxycholate (SDC) (Cat. # D6750, Sigma-Aldrich) solution in 1X PBS for 24 h followed by three 30 min 1x PBS rinses and washed in 0.5% (w/v) sodium dodecyl sulfate (SDS) (Cat#: 436143, Sigma-Aldrich) solution in 1X PBS for 4 h. Tissues were rinsed again with 1X PBS three times 30 min each and digested with DNase (0.66 Units/mL, Cat#: D4513, Sigma-Aldrich) in Sorensen’s Digest Buffer solution for 24 h. Finally, the tissues were rinsed for 24 h with deionized water (DI) water and stored in 70% ethanol at 4°C.

    Techniques: Construct, Standard Deviation

    Validation of puromycin incorporation into nascent proteins and Restrictocin A-induced inhibition of protein synthesis during viral entry. ( A ) H1-HeLa cells were incubated with 100 µg/ml α-sarcin or 50 µg/ml Restrictocin A for 6 hr at 37˚C, followed by treatment with cycloheximide for 30 min prior to a 20 min pulse with 20 µg/ml puromycin. Protein lysates were harvested, resolved by SDS-PAGE and subjected to immunoblotting using antibodies against puromycin and actin. ( B ) H1-HeLa cells were adsorbed for 1 hr with gradient-purified eHAV or naked HAV (~1000 GEs/cell) at 37˚C or with HRV14 (10 PFU/cell) at 33˚C in presence or absence of 50 µg/ml Restrictocin A. The inoculum was removed, cells were rinsed with PBS, and incubated for another 5 hr. Cells were then pulsed with puromycin for 20 min and protein lysates were harvested, resolved by SDS-PAGE, and subjected to immublobotting using antibodies against puromycin and actin. Band intensities were quantified, normalized to loading control, and presented as a percentage of virus plus α-sarcin relative to virus alone. Alternatively, viruses were incubated overnight at 4˚C with human ‘JC’ plasma containing neutralizing HAV antibodies and inoculated as such.

    Journal: eLife

    Article Title: Cellular entry and uncoating of naked and quasi-enveloped human hepatoviruses

    doi: 10.7554/eLife.43983

    Figure Lengend Snippet: Validation of puromycin incorporation into nascent proteins and Restrictocin A-induced inhibition of protein synthesis during viral entry. ( A ) H1-HeLa cells were incubated with 100 µg/ml α-sarcin or 50 µg/ml Restrictocin A for 6 hr at 37˚C, followed by treatment with cycloheximide for 30 min prior to a 20 min pulse with 20 µg/ml puromycin. Protein lysates were harvested, resolved by SDS-PAGE and subjected to immunoblotting using antibodies against puromycin and actin. ( B ) H1-HeLa cells were adsorbed for 1 hr with gradient-purified eHAV or naked HAV (~1000 GEs/cell) at 37˚C or with HRV14 (10 PFU/cell) at 33˚C in presence or absence of 50 µg/ml Restrictocin A. The inoculum was removed, cells were rinsed with PBS, and incubated for another 5 hr. Cells were then pulsed with puromycin for 20 min and protein lysates were harvested, resolved by SDS-PAGE, and subjected to immublobotting using antibodies against puromycin and actin. Band intensities were quantified, normalized to loading control, and presented as a percentage of virus plus α-sarcin relative to virus alone. Alternatively, viruses were incubated overnight at 4˚C with human ‘JC’ plasma containing neutralizing HAV antibodies and inoculated as such.

    Article Snippet: The virus was concentrated by ultracentrifugation at 100,000 ×g for 60 min at 4˚C, and the resulting pellet was resuspended in 250 µl phosphate buffer saline (PBS) and loaded on top of a five-step gradient of 8% to 40% iodixanol (OptiPrep, Sigma) and centrifuged at 165,915 ×g (37,000 rpm) for 24 hr at 4˚C in a Beckman SW55i rotor using a Beckman Optima LE-80K ultracentrifuge.

    Techniques: Inhibition, Incubation, SDS Page, Purification

    Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) AcOSu, PBS (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .

    Journal: Angewandte Chemie (International ed. in English)

    Article Title: Controlled Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides

    doi: 10.1002/anie.201800387

    Figure Lengend Snippet: Enzymatic N -sulfation and chemical acetylation of hexasaccharides. Reagents and conditions: a) 1. AG 50W-X8 resin (pyridinium form); 2. DMSO/H 2 O (95:5), 60% for 23 , 80% for 24 , 72% for 25 ; b) PAPS, NST, MES (50 mM, pH 7.1); c) AcOSu, PBS (pH 7.4), 65% for 29 , 45% for 30 , 100% for 31 .

    Article Snippet: The compounds 26 – 28 were chemically N-acetylated with N -acetyl succinimide (AcOSu) in PBS buffer to give 29 – 31 , respectively, after purification by BioRad P4 size exclusion column chromatography.

    Techniques: Papanicolaou Stain