live dead viability cytotoxicity kit Search Results


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  • 99
    Thermo Fisher live dead baclight bacterial viability kit
    Enhancement of the biofilm growth of isolate 15 by oxacillin at the highest achievable serum concentration* *: As isolate 15 and 22 showed similar responses, only the results for isolate 15 are shown. a and c: stained with Alexa Fluor 555 conjugated wheat germ agglutinin; red signal indicates the presence of polysaccharide. b and d: stained with <t>Live/Dead</t> <t>BacLight</t> bacterial viability kit; green signal indicates the presence of live cells and red signal indicates the presence of dead cells.
    Live Dead Baclight Bacterial Viability Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher live dead viability cytotoxicity kit
    a) Representative confocal microscopy images of RAW 264.7 macrophages incubated with different concentrations of unmodified CNC, NOTA-CNC-Cy5, and DFO-CNC-Cy5 for 96 h. Nuclei of <t>dead</t> cells stain red with EthD-1 after loss off cell membrane integrity, while <t>live</t> cells stain green with Calcein AM due to intracellular esterase activity. Medium and 0.1% saponin are used as controls for live and dead cells, respectively. Scale bar 100 μm. Quantification of the proportion of Calcein AM–positive cells at 6 h (b), 24 h (c), and 96 h (d) of incubation with different concentrations of unmodified and modified cellulose nanocrystals. Columns denote mean ± SD of n = 6.5 ± 0.5 (mean ± SEM) analyzed images per time point. Statistical significance of the difference against the positive control was determined by unpaired Mann-Whitney U test with * p
    Live Dead Viability Cytotoxicity Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 7340 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable aqua dead cell stain kit
    DC subsets are altered and are involved in iNKT activation in both murine and human NASH. ( A ) Representative contour plots of pDC in liver of B6 mice fed ND or CDAA diet. pDC cells were identified as double PDCA-1- and Siglec H-positive cells (PDCA-1 + Siglec H + ) in the <t>live</t> + CD3 − CD45 + CD11c + CD11b − B220 + gate. Numbers on plots indicate the percentage of pDC. Bar graph shows the percentage of pDC in B6 mice fed ND ( n =8) or CDAA diet ( n =6). ( B ) Representative contour plots of the two major cDC subsets, CD103 + cDC and CD11b + cDC, in liver of B6 mice fed ND or CDAA diet. The CD103 + cDC subset was identified as CD103-positive and simultaneously CD11b-negative cells (CD103 + /CD11b − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. The CD11b + cDC subset was identified as CD11b-positive and simultaneously CD103-negative cells (CD11b + /CD103 − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. Numbers on plots indicate the percentage of both subsets. Bar graphs show the percentage of CD103 + cDC (left) and CD11b + cDC (right) in the indicated groups. Each group included 3 mice. ( C ) Histogram shows representative CD1d expression on gated pDC cells in liver of B6 mice fed ND or CDAA diet. ( D ) Representative contour plots of pDC cells in liver of BDCA2-DTR transgenic mice (Tg + ) and littermates (Tg − ) on CDAA diet after DT administration. Bar graph shows the percentage of iNKT cells (αGalCer/CD1d tetramer + ) in liver of Tg − ( n =3) and Tg + ( n =3) mice after DT treatment. All data presented in (A), (B) and (D) are presented as mean ± SEM and are representative of three independent experiments. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001 by unpaired two-sample t-test; ns, not significant. ( E ) Representative dot plots of gating strategy to identify DC subsets in human PBMC. DC cells were gated based on FSC and SSC including both lymphocytes and monocytes. Doublets and <t>dead</t> cells were excluded. (I) CD45 + cells were selected on gated live singlet cells. (II) Lineage (CD3/19/56/14) negative cells were gated on CD45 + cells. (III) Based on CD11c and CD123 expression, pDC were identified as CD123 + CD11c − cells while mDC were identified as CD11c + CD123 − cells. (IV) Based on CD141 and CD1c expression, mDCs were divided in CD141 hi cells or CD1c + cells. (V) The pDC lineage was confirmed by expression of CD303. ( F ) Scatter graphs show the percentage of pDC (left), CD141 hi mDC (center) and CD1c + mDC (right) in PBMC of healthy controls ( n =5) and NASH patients ( n =7). Each point represents an individual. All data are presented as mean ± SEM. *p≤ 0.05 by Mann–Whitney U test.
    Live Dead Fixable Aqua Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable dead cell stain kit
    Flow-based measurement of ADCC activity using annexin V (AnV) staining of target <t>cells.</t> Uncoated CEM cells (unCEM) labeled with CFSE were mixed 1:1 with rgp120-coated CEM cells (cCEM) labeled with CFSE and PKH26 prior to the addition of opsonizing antibodies to be used as target (T) cells. Peripheral blood mononuclear cells (PBMCs) were used as effector (E) cells and mixed at an E:T ratio of 30:1. After 1 h of incubation, cells were <t>stained</t> with annexin V (AnV) and <t>Live/Dead</t> (LD) reagents to quantify the frequency of early/late apoptotic (AnV + ) and dead (LD + ) target CEM cells, respectively, by flow cytometry. (A) Gating strategy. Combined PBMC, cCEM cells, and unCEM cells were gated on by f orward sc atter A (FSC-A) and s ide sc atter A (SSC-A). The frequencies (%) of AnV + and LD + CEM cells were evaluated among cCEM cells (CFSE + PKH26 + , upper right panel) and unCEM cells (CFSE + PKH26 − ; lower right panel) cells opsonized with either 1.5 μg/ml (middle) or 15 μg/ml (right) of HIV + IgG compared to no antibody (No Ig, left). Percentages of AnV + and/or LD + cells are indicated in each quadrant. (B) Dose-response curves showing ADCC activity (% ADCC) as measured by the percentage of AnV + (black symbols) or LD + (gray symbols) cCEM target cells opsonized with either HIV + IgG (filled symbols) or HIV − IgG (empty symbols) after background (No Ab) subtraction. Error bars indicate the standard deviation (s.d.) of replicates and significance was determined by comparing the percentages of ADCC between HIV + IgG and HIV − IgG for all IgG concentrations using AnV or LD (*, P
    Live Dead Fixable Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable violet dead cell stain kit
    Virally-induced IFN-β expression augments DOX-induced apoptosis associated with increased surface exposure of surface CRT, phagocytosis of tumor <t>cell</t> debris by BM-derived DCs, and immunogenicity. Cell death in ID8-R tumor cells treated with OVV-Fc (MOI = 1), DOX (1 µmol/l) or OVV-Fc followed by DOX (12 hours after infection) was determined by staining with Annexin V-FITC and <t>LIVE/DEAD</t> <t>fixable</t> <t>violet</t> to measure the induction of early apoptosis (Annexin V + /LIVE/DEAD fixable violet − ) and late apoptosis/necrosis (Annexin V +/− /LIVE/DEAD fixable violet + ) by flow cytometry 24 hours later. ( a ) One representative experiment of three independent experiments performed is shown. ( b ) Results are presented as the mean ± SD of three independent experiments. * P
    Live Dead Fixable Violet Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2209 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead kit
    SpyA promotes cell death and caspase-3 activation in a manner independent of MAPK and NF-κB signaling. (A) <t>LIVE/DEAD</t> staining illustrating BMDM viability after 4 h of GAS (AP) infection. Cells were infected at an MOI of ~25 or remained uninfected (UI). Scale bar, 100 µM. (B) Numbers of dead cells were counted from multiple field of views ( n = 12). (C) Relative levels of LDH release (percentages compared to uninfected [UI] cells). Δs pyA -infected BMDMs exhibit reduced 2-h cell cytotoxicity at an MOI of 10 or 25. Infection with the SpyA-complemented strain restored LDH release to the WT level ( n = 4). Error bars, SEM. *, P
    Live Dead Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable near ir dead cell stain kit
    Dj-1 depletion inhibits nTreg proliferation and iTreg differentiation via PDH a , Representative histogram overlay of FOXP3 expression, cell proliferation (as labelled by celltrace violet [CTV]) or cell survival as measured by <t>live/dead</t> staining among CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days treated with PDH inhibitor (CPI-613) or control vehicle or without stimulation. b , Percentages of FOXP3 expression among DJ-1 KO or WT CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days. c , Representative flow-cytometry plots of FOXP3 and CTV staining among DJ-1 KO or WT CD4 cells differentiated from Th0 cells under the iTreg differentiation stimulated condition for 3 days. d , Representative flow-cytometry plots of FOXP3 expression among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. e , Representative flow-cytoemtry plots of FOXP3 and CTV staining among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. f , Percentage of living cells under the iTreg differentiation condition for 3 days with different doses of CPI-613 or DCA or control vehicle. g , Representative histogram overlay of FOXP3 expression differentiated from Th0 cells treated with CPI-613 or DCA or control vehicle. The percentages of FOXP3 expressing cells among gated living CD4 T cells were marked. Results represent four ( a, d, e ), three ( f, g ) and two ( b, c ) independent experiments. Data are mean± s.d. The P-values are determined by a two-tailed Student’s t -test. ns or unlabeled, not significant, *P
    Live Dead Fixable Near Ir Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1630 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead assay kit
    Viability staining of MSCs in HA hydrogel constructs after 28 days of in vitro culture for groups containing microspheres with TGF-β3 (MS+T), containing empty microspheres either in chondrogenic media (MS-T+CM) or in standard growth media (MS-T-CM), or with TGFβ-3 alone (T only). Green: <t>live</t> cells; Red: <t>dead</t> cells; scale bar = 100 μm.
    Live Dead Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead viability cytotoxicity assay kit
    Assessment of murine islet viability and function 24 hrs following coating assembly. Coated islets were treated with NHS-PEG-N 3 /4armPEG-MDT/Alg-N 3 , while control islets were exposed to washing steps but not the polymers. Representative <t>live/dead</t> multi-slice projection confocal microscopy images of control (A) and coated (B) islets; (green=viable; red=dead). Metabolic activity, per MTT metabolic assay, (C) and glucose stimulated insulin secretion (GSIS) (D) of murine islets indicate high cytocompatibility of coating. *P
    Live Dead Viability Cytotoxicity Assay Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 997 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable yellow dead cell stain kit
    Interferon regulatory factor 5 (IRF5) is required for toll-like receptor 9/B cell receptor -induced antibody secreting cell differentiation. Isolated human naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and mock-stimulated or stimulated with the indicated cocktails. (A) IRF5 transcript expression was quantified through qPCR on RNA isolated 48 h postnucleofection and 12 h poststimulation with anti-IgM + CpG-B (two-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (B) Protein lysates were prepared from nucleofected B cells 72 h postnucleofection and 24 h poststimulation with anti-IgM + CpG-B. Western blot is one representative experiment out of three performed on n = 3 independent donors. (C) Representative histograms of IRF5 expression 48 h postnucleofection. Viable cells were analyzed through <t>live/dead</t> staining discrimination. (D) Plotted MFI of IRF5 from (C) (one-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (E) Plotted percentage of B cell viability assessed 72 h post-nucleofection, as determined through trypan blue exclusion. Data are from n = 3 independent donors. (F) Representative dot plots from B cell apoptosis quantified following staining with Annexin V and 7 amino-actinomycin D (7-AAD). Early apoptotic events are characterized as Annexin V + 7AAD − , whereas late apoptotic events are Annexin V + 7AAD + . Quantitation is shown in (G) . (G) Average apoptotic B cells from (F) 96 h post-nucleofection and 48 h post-stimulation (Two-way ANOVA with Tukey’s post hoc test; n = 3 independent donors). (H) Isolated naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and stimulated with either CD40L or the combination of CD40L, IL21, anti-IgM, and CpG-B for 7 days. Plasmablast differentiation was quantified through gating of CD19 + CD20 + IgD − CD27 + CD38 + B cells; final CD27 + CD38 + gating is shown. Flow cytometry contour plots are representative of one experiment from a single donor. (I) Average number of plasmablasts from (H) following culture for 7 days (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). (J) Average number of IgD + CD38 lo B cells from (H) following stimulation (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). Error bars represent SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
    Live Dead Fixable Yellow Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 792 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead sperm viability kit
    The sperm viability, as assessed by a fluorescent staining method. <t>Live</t> sperm cells fluoresced green (SYBR-14 dye), and <t>dead</t> sperm cells fluoresced red (PI) (1600×). (A) Control live sperm appeared green, confirming the utility of the assay. The spermatozoa treated with 0.20 mM N-9 (B) and 0.20 mM PD (C) both exhibited PI staining (red). (D) The sperm count showed that 0.20 mM PD killed almost all of the sperm, while 0.20 mM of N-9 only killed 65.5% of the spermatozoa. Each bar represents the mean ± SEM of six sets of observations; *: Significantly different from the control, p
    Live Dead Sperm Viability Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable red dead cell stain kit
    Spermatozoa with loosely attached particles after the washing steps of the fluorescent labelling with <t>LIVE/DEAD</t> Fixable red probe. Confocal laser scanning microscopy
    Live Dead Fixable Red Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher filmtracer live dead biofilm viability kit
    HBD3-C15 and Nystatin (Nys) showed fungicidal activity against C. albicans . C. albicans (6 × 10 6 cells/mL) was incubated for 48 hr on cover glass and treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, 300 µg/mL), aqueous calcium hydroxide (CH, 100 µg/mL), and Nys (20 µg/mL) for 7 days. (a) <t>FilmTracer</t> <t>LIVE/DEAD</t> <t>Biofilm</t> Viability staining was performed and observed under CLSM; (b) The numbers of dead cells (PI stained red) were increased in the group of HBD3-C15 in a dose-dependent manner; (c) The dead cell biomass were significantly higher in the groups of HBD3-C15 (≥ 100 µg/mL) and Nys than CH group ( p
    Filmtracer Live Dead Biofilm Viability Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher live dead fixable far red dead cell stain kit
    Phloxine B staining reflects percentage of dead cells. (A) Example of colony redness score extraction by pyphe-quantify in redness mode. From the acquired input image (i), colors are enhanced and the background subtracted (ii), colonies are identified by local thresholding (iii), and redness is quantified and annotated in the original image (iv). (B) Representative cells for alive, dead and lysed cells using imaging flow cytometry (ImageStream) analysis. Lysed cells show no signal in either the phloxine B or <t>LIVE/DEAD</t> channels. Live cells show an intermediate signal intensity in the phloxine B channel but no LIVE/DEAD signal. Dead cells are brightly stained in both channels. (C) Histogram of intensities in phloxine B channel across 23 samples with three populations (lysed, alive and dead) clearly resolved. (D) Fraction of live cells (live/(lysed+dead)) by ImageStream correlate with colony redness scores (corrected by row/median column normalisation) obtained with pyphe. (E) Co-localisation of phloxine B stain with LIVE/DEAD stain for the standard lab strain 972 . (F) Comparison of Phloxine B staining with LIVE/DEAD stain by ImageStream. Both readouts agree with 99.3% accuracy using the illustrated thresholds.
    Live Dead Fixable Far Red Dead Cell Stain Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Enhancement of the biofilm growth of isolate 15 by oxacillin at the highest achievable serum concentration* *: As isolate 15 and 22 showed similar responses, only the results for isolate 15 are shown. a and c: stained with Alexa Fluor 555 conjugated wheat germ agglutinin; red signal indicates the presence of polysaccharide. b and d: stained with Live/Dead BacLight bacterial viability kit; green signal indicates the presence of live cells and red signal indicates the presence of dead cells.

    Journal: Annals of Clinical Microbiology and Antimicrobials

    Article Title: Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

    doi: 10.1186/1476-0711-9-16

    Figure Lengend Snippet: Enhancement of the biofilm growth of isolate 15 by oxacillin at the highest achievable serum concentration* *: As isolate 15 and 22 showed similar responses, only the results for isolate 15 are shown. a and c: stained with Alexa Fluor 555 conjugated wheat germ agglutinin; red signal indicates the presence of polysaccharide. b and d: stained with Live/Dead BacLight bacterial viability kit; green signal indicates the presence of live cells and red signal indicates the presence of dead cells.

    Article Snippet: Bacterial attachment and viability was then assayed with the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes Inc., Eugene, OR), by adding 200 μl of a mixed solution of SYTO 9 (5 μM) (staining living cells) and propidium iodide (PI, 30 μM) (staining dead cells) into each well, and incubating the microplate at room temperature in the dark for 15 min.

    Techniques: Concentration Assay, Staining

    a Cell disposition of cultures of the strain CECT 8546 of Komagataeibacter europaeus after 16 h with the GqqA protein or BSA visualized by epifluorescence microscopy with the Live/Dead BacLight Kit. b Turbidity measurements at 600 nm during the growth of the strain CECT 8546 of Komagataeibacter europaeus at different concentrations of the GqqA protein: 20 μg/ml ( black solid line ), 10 μg/ml ( gray dotted line ), 5 μg/ml ( black dotted line ), and 5 μg/ml of BSA as a control ( gray solid line ). c ) Glucose decrease in the medium GY during the growth of the strain CECT 8546 of Komagataeibacter europaeus with either 20 μg/ml of GqqA protein ( gray line ) or BSA ( black line ). Error bars represent standard deviations. Values are the mean of three measurements

    Journal: Microbial Cell Factories

    Article Title: GqqA, a novel protein in Komagataeibacter europaeus involved in bacterial quorum quenching and cellulose formation

    doi: 10.1186/s12934-016-0482-y

    Figure Lengend Snippet: a Cell disposition of cultures of the strain CECT 8546 of Komagataeibacter europaeus after 16 h with the GqqA protein or BSA visualized by epifluorescence microscopy with the Live/Dead BacLight Kit. b Turbidity measurements at 600 nm during the growth of the strain CECT 8546 of Komagataeibacter europaeus at different concentrations of the GqqA protein: 20 μg/ml ( black solid line ), 10 μg/ml ( gray dotted line ), 5 μg/ml ( black dotted line ), and 5 μg/ml of BSA as a control ( gray solid line ). c ) Glucose decrease in the medium GY during the growth of the strain CECT 8546 of Komagataeibacter europaeus with either 20 μg/ml of GqqA protein ( gray line ) or BSA ( black line ). Error bars represent standard deviations. Values are the mean of three measurements

    Article Snippet: In addition, epifluorescence microscopy was used to compare the evolution in the growth of this strain after 16 h. A volume of 10 μl from each sample was stained with 1 μl of SYTO9 dye and 1 μl of propidium iodide (PI) dye from the Live/Dead BacLight Kit (Molecular Probes, Eugene, OR, USA).

    Techniques: Epifluorescence Microscopy

    Confocal images showing S. aureus biofilm on the surfaces of the control polyurethane (A) and (+)-usnic acid-loaded polyurethane (B) disks 3 days postinoculation. The grey arrow indicates a cluster of cocci, and the white arrow indicates the biofilm slime matrix. Bar, 55 μm. S. aureus adhered to the usnic acid-loaded polyurethane disk after 30 min (C) and 24 h (D) stained with the Live/Dead BacLight viability kit. Bar, 125 μm. The predominance of red cells after 24 h indicated a progressive loss of viability.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Usnic Acid, a Natural Antimicrobial Agent Able To Inhibit Bacterial Biofilm Formation on Polymer Surfaces

    doi: 10.1128/AAC.48.11.4360-4365.2004

    Figure Lengend Snippet: Confocal images showing S. aureus biofilm on the surfaces of the control polyurethane (A) and (+)-usnic acid-loaded polyurethane (B) disks 3 days postinoculation. The grey arrow indicates a cluster of cocci, and the white arrow indicates the biofilm slime matrix. Bar, 55 μm. S. aureus adhered to the usnic acid-loaded polyurethane disk after 30 min (C) and 24 h (D) stained with the Live/Dead BacLight viability kit. Bar, 125 μm. The predominance of red cells after 24 h indicated a progressive loss of viability.

    Article Snippet: Bacteria were then stained using a Live/Dead BacLight viability kit (Molecular Probes).

    Techniques: Staining

    Effect of CDA combined antimicrobial treatments on killing of pre-established biofilms. CDA treatment reverses biofilm formation in pre-established biofilms and cells remaining on the surface are easily removed and killed various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment, then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+CDA) for 1 h and stained with LIVE/DEAD staining to allow analysis using fluorescence microscopy. The images show microscopic pictures of the biofilms on the surface of cover slip after combinatorial treatments. Images are top-down views (x-y plane); scale bars: 50 µm. Results are representative of 3 separate experiments.

    Journal: PLoS ONE

    Article Title: Unsaturated Fatty Acid, cis-2-Decenoic Acid, in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

    doi: 10.1371/journal.pone.0101677

    Figure Lengend Snippet: Effect of CDA combined antimicrobial treatments on killing of pre-established biofilms. CDA treatment reverses biofilm formation in pre-established biofilms and cells remaining on the surface are easily removed and killed various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment, then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+CDA) for 1 h and stained with LIVE/DEAD staining to allow analysis using fluorescence microscopy. The images show microscopic pictures of the biofilms on the surface of cover slip after combinatorial treatments. Images are top-down views (x-y plane); scale bars: 50 µm. Results are representative of 3 separate experiments.

    Article Snippet: After 1 h treatment, biofilms were stained with a LIVE/DEAD Bac Light bacterial viability kit (Molecular Probes).

    Techniques: Flow Cytometry, Staining, Fluorescence, Microscopy

    Effect of CDA combined disinfectant or antibiotic treatments on biofilms surface area. Following dispersion of biofilms by CDA, cells remaining on the surface are easily killed and removed by various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment and then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+ CDA) for 1 h, stained with LIVE/DEAD staining and quantified (percent surface coverage) using digital image analysis. The bars show the levels of biofilm biomass after treatment with antimicrobials alone or combined with 310 nM CDA. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value

    Journal: PLoS ONE

    Article Title: Unsaturated Fatty Acid, cis-2-Decenoic Acid, in Combination with Disinfectants or Antibiotics Removes Pre-Established Biofilms Formed by Food-Related Bacteria

    doi: 10.1371/journal.pone.0101677

    Figure Lengend Snippet: Effect of CDA combined disinfectant or antibiotic treatments on biofilms surface area. Following dispersion of biofilms by CDA, cells remaining on the surface are easily killed and removed by various disinfectants (Epimax S and Percidine) or antibiotics (vancomycin; Van, ampicillin; Amp, ciprofloxacin; Cip) in biofilms grown in continuous culture flow cells. Pre-established biofilms were grown for 48 h without any treatment and then were treated with indicated concentrations of antimicrobials alone (- CDA) or combined with 310 nM CDA (+ CDA) for 1 h, stained with LIVE/DEAD staining and quantified (percent surface coverage) using digital image analysis. The bars show the levels of biofilm biomass after treatment with antimicrobials alone or combined with 310 nM CDA. Error bars indicate standard errors (n = 3) and mean values sharing at least one common lowercase letter shown above the bars are not significantly different ( P-value

    Article Snippet: After 1 h treatment, biofilms were stained with a LIVE/DEAD Bac Light bacterial viability kit (Molecular Probes).

    Techniques: Flow Cytometry, Staining

    a) Representative confocal microscopy images of RAW 264.7 macrophages incubated with different concentrations of unmodified CNC, NOTA-CNC-Cy5, and DFO-CNC-Cy5 for 96 h. Nuclei of dead cells stain red with EthD-1 after loss off cell membrane integrity, while live cells stain green with Calcein AM due to intracellular esterase activity. Medium and 0.1% saponin are used as controls for live and dead cells, respectively. Scale bar 100 μm. Quantification of the proportion of Calcein AM–positive cells at 6 h (b), 24 h (c), and 96 h (d) of incubation with different concentrations of unmodified and modified cellulose nanocrystals. Columns denote mean ± SD of n = 6.5 ± 0.5 (mean ± SEM) analyzed images per time point. Statistical significance of the difference against the positive control was determined by unpaired Mann-Whitney U test with * p

    Journal: Nuclear medicine and biology

    Article Title: Multimodality labeling strategies for the investigation of nanocrystalline cellulose biodistribution in a mouse model of breast cancer

    doi: 10.1016/j.nucmedbio.2019.11.002

    Figure Lengend Snippet: a) Representative confocal microscopy images of RAW 264.7 macrophages incubated with different concentrations of unmodified CNC, NOTA-CNC-Cy5, and DFO-CNC-Cy5 for 96 h. Nuclei of dead cells stain red with EthD-1 after loss off cell membrane integrity, while live cells stain green with Calcein AM due to intracellular esterase activity. Medium and 0.1% saponin are used as controls for live and dead cells, respectively. Scale bar 100 μm. Quantification of the proportion of Calcein AM–positive cells at 6 h (b), 24 h (c), and 96 h (d) of incubation with different concentrations of unmodified and modified cellulose nanocrystals. Columns denote mean ± SD of n = 6.5 ± 0.5 (mean ± SEM) analyzed images per time point. Statistical significance of the difference against the positive control was determined by unpaired Mann-Whitney U test with * p

    Article Snippet: Cytotoxicity and confocal microscopy in murine RAW 264.7 macrophages The cytotoxicity of the modified CNC probes was determined in RAW 264.7 macrophages using the Molecular Probes LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.

    Techniques: Confocal Microscopy, Incubation, Staining, Ethidium Homodimer Assay, Activity Assay, Modification, Positive Control, MANN-WHITNEY

    DC subsets are altered and are involved in iNKT activation in both murine and human NASH. ( A ) Representative contour plots of pDC in liver of B6 mice fed ND or CDAA diet. pDC cells were identified as double PDCA-1- and Siglec H-positive cells (PDCA-1 + Siglec H + ) in the live + CD3 − CD45 + CD11c + CD11b − B220 + gate. Numbers on plots indicate the percentage of pDC. Bar graph shows the percentage of pDC in B6 mice fed ND ( n =8) or CDAA diet ( n =6). ( B ) Representative contour plots of the two major cDC subsets, CD103 + cDC and CD11b + cDC, in liver of B6 mice fed ND or CDAA diet. The CD103 + cDC subset was identified as CD103-positive and simultaneously CD11b-negative cells (CD103 + /CD11b − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. The CD11b + cDC subset was identified as CD11b-positive and simultaneously CD103-negative cells (CD11b + /CD103 − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. Numbers on plots indicate the percentage of both subsets. Bar graphs show the percentage of CD103 + cDC (left) and CD11b + cDC (right) in the indicated groups. Each group included 3 mice. ( C ) Histogram shows representative CD1d expression on gated pDC cells in liver of B6 mice fed ND or CDAA diet. ( D ) Representative contour plots of pDC cells in liver of BDCA2-DTR transgenic mice (Tg + ) and littermates (Tg − ) on CDAA diet after DT administration. Bar graph shows the percentage of iNKT cells (αGalCer/CD1d tetramer + ) in liver of Tg − ( n =3) and Tg + ( n =3) mice after DT treatment. All data presented in (A), (B) and (D) are presented as mean ± SEM and are representative of three independent experiments. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001 by unpaired two-sample t-test; ns, not significant. ( E ) Representative dot plots of gating strategy to identify DC subsets in human PBMC. DC cells were gated based on FSC and SSC including both lymphocytes and monocytes. Doublets and dead cells were excluded. (I) CD45 + cells were selected on gated live singlet cells. (II) Lineage (CD3/19/56/14) negative cells were gated on CD45 + cells. (III) Based on CD11c and CD123 expression, pDC were identified as CD123 + CD11c − cells while mDC were identified as CD11c + CD123 − cells. (IV) Based on CD141 and CD1c expression, mDCs were divided in CD141 hi cells or CD1c + cells. (V) The pDC lineage was confirmed by expression of CD303. ( F ) Scatter graphs show the percentage of pDC (left), CD141 hi mDC (center) and CD1c + mDC (right) in PBMC of healthy controls ( n =5) and NASH patients ( n =7). Each point represents an individual. All data are presented as mean ± SEM. *p≤ 0.05 by Mann–Whitney U test.

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Differential activation of hepatic iNKT cell subsets plays a key role in progression of nonalcoholic steatohepatitis

    doi: 10.4049/jimmunol.1800614

    Figure Lengend Snippet: DC subsets are altered and are involved in iNKT activation in both murine and human NASH. ( A ) Representative contour plots of pDC in liver of B6 mice fed ND or CDAA diet. pDC cells were identified as double PDCA-1- and Siglec H-positive cells (PDCA-1 + Siglec H + ) in the live + CD3 − CD45 + CD11c + CD11b − B220 + gate. Numbers on plots indicate the percentage of pDC. Bar graph shows the percentage of pDC in B6 mice fed ND ( n =8) or CDAA diet ( n =6). ( B ) Representative contour plots of the two major cDC subsets, CD103 + cDC and CD11b + cDC, in liver of B6 mice fed ND or CDAA diet. The CD103 + cDC subset was identified as CD103-positive and simultaneously CD11b-negative cells (CD103 + /CD11b − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. The CD11b + cDC subset was identified as CD11b-positive and simultaneously CD103-negative cells (CD11b + /CD103 − ) in the live + CD3 − CD19 − CD45 + MHC-II + CD11c hi gate. Numbers on plots indicate the percentage of both subsets. Bar graphs show the percentage of CD103 + cDC (left) and CD11b + cDC (right) in the indicated groups. Each group included 3 mice. ( C ) Histogram shows representative CD1d expression on gated pDC cells in liver of B6 mice fed ND or CDAA diet. ( D ) Representative contour plots of pDC cells in liver of BDCA2-DTR transgenic mice (Tg + ) and littermates (Tg − ) on CDAA diet after DT administration. Bar graph shows the percentage of iNKT cells (αGalCer/CD1d tetramer + ) in liver of Tg − ( n =3) and Tg + ( n =3) mice after DT treatment. All data presented in (A), (B) and (D) are presented as mean ± SEM and are representative of three independent experiments. *p≤ 0.05, **p≤ 0.01, ***p≤ 0.001 by unpaired two-sample t-test; ns, not significant. ( E ) Representative dot plots of gating strategy to identify DC subsets in human PBMC. DC cells were gated based on FSC and SSC including both lymphocytes and monocytes. Doublets and dead cells were excluded. (I) CD45 + cells were selected on gated live singlet cells. (II) Lineage (CD3/19/56/14) negative cells were gated on CD45 + cells. (III) Based on CD11c and CD123 expression, pDC were identified as CD123 + CD11c − cells while mDC were identified as CD11c + CD123 − cells. (IV) Based on CD141 and CD1c expression, mDCs were divided in CD141 hi cells or CD1c + cells. (V) The pDC lineage was confirmed by expression of CD303. ( F ) Scatter graphs show the percentage of pDC (left), CD141 hi mDC (center) and CD1c + mDC (right) in PBMC of healthy controls ( n =5) and NASH patients ( n =7). Each point represents an individual. All data are presented as mean ± SEM. *p≤ 0.05 by Mann–Whitney U test.

    Article Snippet: Human antibodies were as follow: anti-CD3 (UCHT1), anti-CD19 (SJ25-C1), anti-CD4 (RPA-T4), anti-CD123 (CD1d42), anti-CD45 (HI30) from BD; anti-Vα24-Jα18 TCR (6B11) from eBioscience; anti-CXCR3 (49801) from R & D Systems; anti-CD14 (HCD14), anti-CD56 (5.1H11), anti-CD11c (Bu15), anti-CD303 (201A), anti-CD141 (M80), anti-CD1c (ICRF44) from BioLegend and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit from Thermo Fisher Scientific.

    Techniques: Activation Assay, Mouse Assay, Expressing, Transgenic Assay, MANN-WHITNEY

    A cell-cell dependent crosstalk between tumoral MDSCs and Treg cells. (A) Purified CD4 + T cells or Treg cells (CD4 + CD25 + ) from TB mice were cocultured with tumoral MDSCs. The percentage of CD4 + Foxp3 + cells was evaluated by flow cytometry after 4 days of culture in the presence of CD3/CD28 stimulation. (B) Purified CD4 + T cells from TB mice were cocultured with tumoral MDSCs at 1:3 ratio in conventional dishes or using Transwell chamber to separate the 2 cell populations. The percentage of CD4 + Foxp3 + cells was evaluated after 4 days by flow cytometry. (C) Purified Treg cells (CD4 + CD25 + ) from spleen of tumor-bearing mice (Treg STB) or tumor (Treg PTB) were cocultured with tumoral MDSCs for 24 or 48 h. The viability of MDSCs was then assessed by flow cytometry using LIVE / DEAD Fixable Aqua Dead stain.

    Journal: Frontiers in Immunology

    Article Title: Deciphering the Crosstalk Between Myeloid-Derived Suppressor Cells and Regulatory T Cells in Pancreatic Ductal Adenocarcinoma

    doi: 10.3389/fimmu.2019.03070

    Figure Lengend Snippet: A cell-cell dependent crosstalk between tumoral MDSCs and Treg cells. (A) Purified CD4 + T cells or Treg cells (CD4 + CD25 + ) from TB mice were cocultured with tumoral MDSCs. The percentage of CD4 + Foxp3 + cells was evaluated by flow cytometry after 4 days of culture in the presence of CD3/CD28 stimulation. (B) Purified CD4 + T cells from TB mice were cocultured with tumoral MDSCs at 1:3 ratio in conventional dishes or using Transwell chamber to separate the 2 cell populations. The percentage of CD4 + Foxp3 + cells was evaluated after 4 days by flow cytometry. (C) Purified Treg cells (CD4 + CD25 + ) from spleen of tumor-bearing mice (Treg STB) or tumor (Treg PTB) were cocultured with tumoral MDSCs for 24 or 48 h. The viability of MDSCs was then assessed by flow cytometry using LIVE / DEAD Fixable Aqua Dead stain.

    Article Snippet: Dead cells were excluded using LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen).

    Techniques: Purification, Mouse Assay, Flow Cytometry, Staining

    Flow-based measurement of ADCC activity using annexin V (AnV) staining of target cells. Uncoated CEM cells (unCEM) labeled with CFSE were mixed 1:1 with rgp120-coated CEM cells (cCEM) labeled with CFSE and PKH26 prior to the addition of opsonizing antibodies to be used as target (T) cells. Peripheral blood mononuclear cells (PBMCs) were used as effector (E) cells and mixed at an E:T ratio of 30:1. After 1 h of incubation, cells were stained with annexin V (AnV) and Live/Dead (LD) reagents to quantify the frequency of early/late apoptotic (AnV + ) and dead (LD + ) target CEM cells, respectively, by flow cytometry. (A) Gating strategy. Combined PBMC, cCEM cells, and unCEM cells were gated on by f orward sc atter A (FSC-A) and s ide sc atter A (SSC-A). The frequencies (%) of AnV + and LD + CEM cells were evaluated among cCEM cells (CFSE + PKH26 + , upper right panel) and unCEM cells (CFSE + PKH26 − ; lower right panel) cells opsonized with either 1.5 μg/ml (middle) or 15 μg/ml (right) of HIV + IgG compared to no antibody (No Ig, left). Percentages of AnV + and/or LD + cells are indicated in each quadrant. (B) Dose-response curves showing ADCC activity (% ADCC) as measured by the percentage of AnV + (black symbols) or LD + (gray symbols) cCEM target cells opsonized with either HIV + IgG (filled symbols) or HIV − IgG (empty symbols) after background (No Ab) subtraction. Error bars indicate the standard deviation (s.d.) of replicates and significance was determined by comparing the percentages of ADCC between HIV + IgG and HIV − IgG for all IgG concentrations using AnV or LD (*, P

    Journal: mBio

    Article Title: Antibody-Dependent Cellular Cytotoxicity-Competent Antibodies against HIV-1-Infected Cells in Plasma from HIV-Infected Subjects

    doi: 10.1128/mBio.02690-19

    Figure Lengend Snippet: Flow-based measurement of ADCC activity using annexin V (AnV) staining of target cells. Uncoated CEM cells (unCEM) labeled with CFSE were mixed 1:1 with rgp120-coated CEM cells (cCEM) labeled with CFSE and PKH26 prior to the addition of opsonizing antibodies to be used as target (T) cells. Peripheral blood mononuclear cells (PBMCs) were used as effector (E) cells and mixed at an E:T ratio of 30:1. After 1 h of incubation, cells were stained with annexin V (AnV) and Live/Dead (LD) reagents to quantify the frequency of early/late apoptotic (AnV + ) and dead (LD + ) target CEM cells, respectively, by flow cytometry. (A) Gating strategy. Combined PBMC, cCEM cells, and unCEM cells were gated on by f orward sc atter A (FSC-A) and s ide sc atter A (SSC-A). The frequencies (%) of AnV + and LD + CEM cells were evaluated among cCEM cells (CFSE + PKH26 + , upper right panel) and unCEM cells (CFSE + PKH26 − ; lower right panel) cells opsonized with either 1.5 μg/ml (middle) or 15 μg/ml (right) of HIV + IgG compared to no antibody (No Ig, left). Percentages of AnV + and/or LD + cells are indicated in each quadrant. (B) Dose-response curves showing ADCC activity (% ADCC) as measured by the percentage of AnV + (black symbols) or LD + (gray symbols) cCEM target cells opsonized with either HIV + IgG (filled symbols) or HIV − IgG (empty symbols) after background (No Ab) subtraction. Error bars indicate the standard deviation (s.d.) of replicates and significance was determined by comparing the percentages of ADCC between HIV + IgG and HIV − IgG for all IgG concentrations using AnV or LD (*, P

    Article Snippet: A Live/Dead fixable dead cell stain kit (Invitrogen, St Laurent, QC, Canada) was used to quantify dead cells by flow cytometry.

    Techniques: Flow Cytometry, Activity Assay, Staining, Labeling, Incubation, Cytometry, Standard Deviation

    Kinetics of Ag-specific T-cell survival and proliferation attributable to rhIL-7 A . PBMC were CFSE labelled on d0 of culture, then conditioned with GM+R848+LPS and either unpulsed or pulsed with CAN, then transitioned to recombinant human rhIL-7+rhIL-2 versus rhIL-2 alone beginning on d2 of culture. In this analysis, representative of four biological replicates, T-cell proliferation over time is indicated by the right-to-left-shift as cell division dilutes intracellular CFSE. B . Left panel displays % viable T-cells over time. No significant differences were observed prior to d12, whereas at d12 and d16 viability remained significantly higher for T-cells exposed to CAN, rhIL-7 and rhIL-2 compared to CAN and rhIL-2 without rhIL-7. Right panel demonstrates that the superior viability of the CAN, rhIL-7 and rhIL-2 group from d12 forward corresponded to sustained numeric expansion significantly greater than when rhIL-7 was omitted. Statistical calculations in both panels of B . show averaged results of four biological replicates. C . Fate of individual PBMC constituents during optimized Ag-driven cultures. PBMC were labelled on d0 with Cell Trace Violet (CTV), conditioned with GM+R848+LPS, pulsed with CAN, then transitioned to rhIL-7 plus rhIL-2. Graph shows in log scale the absolute proportions and numbers of PBMC subpopulations over time, distinguishing CD33+ myeloid cells, never proliferated T-cells (undiluted CTV), and already proliferating T-cells (i.e., CTV diluted due to previous cell division). Data are representative of two biological replicates. D . T-cell cultures were labelled with CTV at d9 to facilitate monitoring of continuing proliferation after that time point in conjunction with live-dead staining. Panel representative of two biological replicates displays d16 of culture, with dot plots showing unproliferated dead cells (RUQ), unproliferated live cells (RLQ), proliferated dead cells (LUQ) and proliferated live cells (LLQ). At the d16 timepoint the continuing inclusion of rhIL-7 has licensed sustained high viability B ., D . and proliferation A ., D . resulting in marked net numeric T-cell expansion B ., C . as well as further enrichment of CAN-specific CD4+ and CD8+ T-cells (Figure 2 ).

    Journal: Oncotarget

    Article Title: Surrogate in vitro activation of innate immunity synergizes with interleukin-7 to unleash rapid antigen-driven outgrowth of CD4+ and CD8+ human peripheral blood T-cells naturally recognizing MUC1, HER2/neu and other tumor-associated antigens

    doi: 10.18632/oncotarget.13911

    Figure Lengend Snippet: Kinetics of Ag-specific T-cell survival and proliferation attributable to rhIL-7 A . PBMC were CFSE labelled on d0 of culture, then conditioned with GM+R848+LPS and either unpulsed or pulsed with CAN, then transitioned to recombinant human rhIL-7+rhIL-2 versus rhIL-2 alone beginning on d2 of culture. In this analysis, representative of four biological replicates, T-cell proliferation over time is indicated by the right-to-left-shift as cell division dilutes intracellular CFSE. B . Left panel displays % viable T-cells over time. No significant differences were observed prior to d12, whereas at d12 and d16 viability remained significantly higher for T-cells exposed to CAN, rhIL-7 and rhIL-2 compared to CAN and rhIL-2 without rhIL-7. Right panel demonstrates that the superior viability of the CAN, rhIL-7 and rhIL-2 group from d12 forward corresponded to sustained numeric expansion significantly greater than when rhIL-7 was omitted. Statistical calculations in both panels of B . show averaged results of four biological replicates. C . Fate of individual PBMC constituents during optimized Ag-driven cultures. PBMC were labelled on d0 with Cell Trace Violet (CTV), conditioned with GM+R848+LPS, pulsed with CAN, then transitioned to rhIL-7 plus rhIL-2. Graph shows in log scale the absolute proportions and numbers of PBMC subpopulations over time, distinguishing CD33+ myeloid cells, never proliferated T-cells (undiluted CTV), and already proliferating T-cells (i.e., CTV diluted due to previous cell division). Data are representative of two biological replicates. D . T-cell cultures were labelled with CTV at d9 to facilitate monitoring of continuing proliferation after that time point in conjunction with live-dead staining. Panel representative of two biological replicates displays d16 of culture, with dot plots showing unproliferated dead cells (RUQ), unproliferated live cells (RLQ), proliferated dead cells (LUQ) and proliferated live cells (LLQ). At the d16 timepoint the continuing inclusion of rhIL-7 has licensed sustained high viability B ., D . and proliferation A ., D . resulting in marked net numeric T-cell expansion B ., C . as well as further enrichment of CAN-specific CD4+ and CD8+ T-cells (Figure 2 ).

    Article Snippet: Fluorescently-conjugated mAb targeting cell surface proteins were then added, also including Live/Dead UV Blue Stain to delineate viability (Life Technologies/Thermo-Fisher #L23105).

    Techniques: Recombinant, Staining

    Virally-induced IFN-β expression augments DOX-induced apoptosis associated with increased surface exposure of surface CRT, phagocytosis of tumor cell debris by BM-derived DCs, and immunogenicity. Cell death in ID8-R tumor cells treated with OVV-Fc (MOI = 1), DOX (1 µmol/l) or OVV-Fc followed by DOX (12 hours after infection) was determined by staining with Annexin V-FITC and LIVE/DEAD fixable violet to measure the induction of early apoptosis (Annexin V + /LIVE/DEAD fixable violet − ) and late apoptosis/necrosis (Annexin V +/− /LIVE/DEAD fixable violet + ) by flow cytometry 24 hours later. ( a ) One representative experiment of three independent experiments performed is shown. ( b ) Results are presented as the mean ± SD of three independent experiments. * P

    Journal: Molecular Therapy Oncolytics

    Article Title: Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy

    doi: 10.1038/mto.2016.34

    Figure Lengend Snippet: Virally-induced IFN-β expression augments DOX-induced apoptosis associated with increased surface exposure of surface CRT, phagocytosis of tumor cell debris by BM-derived DCs, and immunogenicity. Cell death in ID8-R tumor cells treated with OVV-Fc (MOI = 1), DOX (1 µmol/l) or OVV-Fc followed by DOX (12 hours after infection) was determined by staining with Annexin V-FITC and LIVE/DEAD fixable violet to measure the induction of early apoptosis (Annexin V + /LIVE/DEAD fixable violet − ) and late apoptosis/necrosis (Annexin V +/− /LIVE/DEAD fixable violet + ) by flow cytometry 24 hours later. ( a ) One representative experiment of three independent experiments performed is shown. ( b ) Results are presented as the mean ± SD of three independent experiments. * P

    Article Snippet: Before specific antibody staining, cells were incubated with Fc blocker (anti-CD16/CD32 mAb) for 10 minutes followed by Live/Dead Fixable Violet Dead Cell stain kit (Thermo Fisher Scientific) to assess live/dead cells, and analyzed on a LRS II flow cytometer (BD Biosciences).

    Techniques: Expressing, Derivative Assay, Infection, Staining, Flow Cytometry, Cytometry

    Effect of the CXCR4-A-Fc fusion protein on ID8-R tumor growth. ( a ) Cell death in ID8-R tumor cells treated with soluble CXCR4-A-Fc fusion protein (100 µg/ml) for 24 hours was determined by staining with Annexin V-FITC and LIVE/DEAD fixable violet. Tumor cells treated with soluble Fc fragment of mouse IgG2a serve as controls. One representative experiment of three independent experiments performed is shown. ( b ) C57BL/6 mice ( n = 8 − 10) were injected i.p. with 2 × 10 5 ID8-R cells. Oncolytic virotherapy with OVV-CXCR4-A-Fc or OVV-Fc (10 8 PFU delivered i.p.) was initiated 10 days later. In parallel experiments, tumor-bearing mice were treated with PLD (10 mg/kg) delivered i.v. or PLD was delivered to virally-treated mice 8 days after virus injection. Control mice were treated with PBS. Tumor progression was monitored by bioluminescence imaging using the Xenogen IVIS Imaging System. Data points represent mean ± SD. ( c ) Kaplan–Meier survival plots were prepared and significance was determined using the log-rank method. * P

    Journal: Molecular Therapy Oncolytics

    Article Title: Reprogramming antitumor immunity against chemoresistant ovarian cancer by a CXCR4 antagonist-armed viral oncotherapy

    doi: 10.1038/mto.2016.34

    Figure Lengend Snippet: Effect of the CXCR4-A-Fc fusion protein on ID8-R tumor growth. ( a ) Cell death in ID8-R tumor cells treated with soluble CXCR4-A-Fc fusion protein (100 µg/ml) for 24 hours was determined by staining with Annexin V-FITC and LIVE/DEAD fixable violet. Tumor cells treated with soluble Fc fragment of mouse IgG2a serve as controls. One representative experiment of three independent experiments performed is shown. ( b ) C57BL/6 mice ( n = 8 − 10) were injected i.p. with 2 × 10 5 ID8-R cells. Oncolytic virotherapy with OVV-CXCR4-A-Fc or OVV-Fc (10 8 PFU delivered i.p.) was initiated 10 days later. In parallel experiments, tumor-bearing mice were treated with PLD (10 mg/kg) delivered i.v. or PLD was delivered to virally-treated mice 8 days after virus injection. Control mice were treated with PBS. Tumor progression was monitored by bioluminescence imaging using the Xenogen IVIS Imaging System. Data points represent mean ± SD. ( c ) Kaplan–Meier survival plots were prepared and significance was determined using the log-rank method. * P

    Article Snippet: Before specific antibody staining, cells were incubated with Fc blocker (anti-CD16/CD32 mAb) for 10 minutes followed by Live/Dead Fixable Violet Dead Cell stain kit (Thermo Fisher Scientific) to assess live/dead cells, and analyzed on a LRS II flow cytometer (BD Biosciences).

    Techniques: Staining, Mouse Assay, Injection, Imaging

    SpyA promotes cell death and caspase-3 activation in a manner independent of MAPK and NF-κB signaling. (A) LIVE/DEAD staining illustrating BMDM viability after 4 h of GAS (AP) infection. Cells were infected at an MOI of ~25 or remained uninfected (UI). Scale bar, 100 µM. (B) Numbers of dead cells were counted from multiple field of views ( n = 12). (C) Relative levels of LDH release (percentages compared to uninfected [UI] cells). Δs pyA -infected BMDMs exhibit reduced 2-h cell cytotoxicity at an MOI of 10 or 25. Infection with the SpyA-complemented strain restored LDH release to the WT level ( n = 4). Error bars, SEM. *, P

    Journal: mBio

    Article Title: A Group A Streptococcus ADP-Ribosyltransferase Toxin Stimulates a Protective Interleukin 1β-Dependent Macrophage Immune Response

    doi: 10.1128/mBio.00133-15

    Figure Lengend Snippet: SpyA promotes cell death and caspase-3 activation in a manner independent of MAPK and NF-κB signaling. (A) LIVE/DEAD staining illustrating BMDM viability after 4 h of GAS (AP) infection. Cells were infected at an MOI of ~25 or remained uninfected (UI). Scale bar, 100 µM. (B) Numbers of dead cells were counted from multiple field of views ( n = 12). (C) Relative levels of LDH release (percentages compared to uninfected [UI] cells). Δs pyA -infected BMDMs exhibit reduced 2-h cell cytotoxicity at an MOI of 10 or 25. Infection with the SpyA-complemented strain restored LDH release to the WT level ( n = 4). Error bars, SEM. *, P

    Article Snippet: Cell viability staining was visualized using a LIVE/DEAD kit for mammalian cells (Invitrogen) following the manufacturer’s protocol.

    Techniques: Activation Assay, Staining, Infection

    Robust cell viability in the 3D adipose construct. ( A – D ) Constructs were processed for Live (calcein, green)/Dead (EthD-1, red) staining after 28 days in culture. ( A , B ) Static cultures; ( C , D ) dynamic cultures (scale bar, 1 mm); ( A , C ) growth medium; and ( B , D ) adipogenic medium. There was increased EthD-1 positive (dead) staining in the dynamic constructs as compared to the static constructs, while no apparent differences were observed between the cultures in the growth medium versus the adipogenic medium. ( E ) The area positive for Calcein staining over a total area demonstrated a significant decrease in the cell viability in dynamic cultures as compared to static cultures (N = 3, ** p

    Journal: Biomolecules

    Article Title: Adipose Tissue-Derived Stem Cells Retain Their Adipocyte Differentiation Potential in Three-Dimensional Hydrogels and Bioreactors †

    doi: 10.3390/biom10071070

    Figure Lengend Snippet: Robust cell viability in the 3D adipose construct. ( A – D ) Constructs were processed for Live (calcein, green)/Dead (EthD-1, red) staining after 28 days in culture. ( A , B ) Static cultures; ( C , D ) dynamic cultures (scale bar, 1 mm); ( A , C ) growth medium; and ( B , D ) adipogenic medium. There was increased EthD-1 positive (dead) staining in the dynamic constructs as compared to the static constructs, while no apparent differences were observed between the cultures in the growth medium versus the adipogenic medium. ( E ) The area positive for Calcein staining over a total area demonstrated a significant decrease in the cell viability in dynamic cultures as compared to static cultures (N = 3, ** p

    Article Snippet: Live/Dead StainingIn order to determine the cell viability after 28 days of culture, the constructs were first washed in Dulbecco’s phosphate buffered saline (DPBS, Gibco) before being stained with a Live/Dead staining kit (ThermoScientific) according to the manufacturer’s protocol.

    Techniques: Construct, Ethidium Homodimer Assay, Staining

    SRVF causes severe cytotoxicity in HT1080 cells. After incubating cells with the indicated concentrations of SR, VF, or SRVF for 24 h, live and dead cells were examined under a fluorescence microscope using the LIVE/DEAD Cell Imaging Kit. Data are expressed as the mean ± SD of five random fields per sample and are representative of three independent experiments.

    Journal: Scientific Reports

    Article Title: SRVF, a novel herbal formula including Scrophulariae Radix and Viticis Fructus, disrupts focal adhesion and causes detachment-induced apoptosis in malignant cancer cells

    doi: 10.1038/s41598-017-12934-y

    Figure Lengend Snippet: SRVF causes severe cytotoxicity in HT1080 cells. After incubating cells with the indicated concentrations of SR, VF, or SRVF for 24 h, live and dead cells were examined under a fluorescence microscope using the LIVE/DEAD Cell Imaging Kit. Data are expressed as the mean ± SD of five random fields per sample and are representative of three independent experiments.

    Article Snippet: Live and dead cell staining To visualize the live and dead cells, LIVE/DEAD Cell Imaging Kit (Invitrogen, Carlsbad, CA, USA) was used.

    Techniques: Fluorescence, Microscopy, Imaging

    Dj-1 depletion inhibits nTreg proliferation and iTreg differentiation via PDH a , Representative histogram overlay of FOXP3 expression, cell proliferation (as labelled by celltrace violet [CTV]) or cell survival as measured by live/dead staining among CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days treated with PDH inhibitor (CPI-613) or control vehicle or without stimulation. b , Percentages of FOXP3 expression among DJ-1 KO or WT CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days. c , Representative flow-cytometry plots of FOXP3 and CTV staining among DJ-1 KO or WT CD4 cells differentiated from Th0 cells under the iTreg differentiation stimulated condition for 3 days. d , Representative flow-cytometry plots of FOXP3 expression among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. e , Representative flow-cytoemtry plots of FOXP3 and CTV staining among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. f , Percentage of living cells under the iTreg differentiation condition for 3 days with different doses of CPI-613 or DCA or control vehicle. g , Representative histogram overlay of FOXP3 expression differentiated from Th0 cells treated with CPI-613 or DCA or control vehicle. The percentages of FOXP3 expressing cells among gated living CD4 T cells were marked. Results represent four ( a, d, e ), three ( f, g ) and two ( b, c ) independent experiments. Data are mean± s.d. The P-values are determined by a two-tailed Student’s t -test. ns or unlabeled, not significant, *P

    Journal: bioRxiv

    Article Title: PARK7/DJ-1 promotes pyruvate dehydrogenase activity and maintains Treg homeostasis

    doi: 10.1101/2019.12.20.884809

    Figure Lengend Snippet: Dj-1 depletion inhibits nTreg proliferation and iTreg differentiation via PDH a , Representative histogram overlay of FOXP3 expression, cell proliferation (as labelled by celltrace violet [CTV]) or cell survival as measured by live/dead staining among CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days treated with PDH inhibitor (CPI-613) or control vehicle or without stimulation. b , Percentages of FOXP3 expression among DJ-1 KO or WT CD4+ cells differentiated from Th0 cells under the iTreg differentiation condition stimulated for 3 days. c , Representative flow-cytometry plots of FOXP3 and CTV staining among DJ-1 KO or WT CD4 cells differentiated from Th0 cells under the iTreg differentiation stimulated condition for 3 days. d , Representative flow-cytometry plots of FOXP3 expression among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. e , Representative flow-cytoemtry plots of FOXP3 and CTV staining among total WT CD4 T cells differentiated from Th0 cells treated with different doses of CPI-613 or control vehicle. f , Percentage of living cells under the iTreg differentiation condition for 3 days with different doses of CPI-613 or DCA or control vehicle. g , Representative histogram overlay of FOXP3 expression differentiated from Th0 cells treated with CPI-613 or DCA or control vehicle. The percentages of FOXP3 expressing cells among gated living CD4 T cells were marked. Results represent four ( a, d, e ), three ( f, g ) and two ( b, c ) independent experiments. Data are mean± s.d. The P-values are determined by a two-tailed Student’s t -test. ns or unlabeled, not significant, *P

    Article Snippet: The viability of cells was assessed by using the LIVE/DEAD® Fixable Near-IR Dead Cell Stain kit (L10119, Thermo Fisher Scientific) (dilution 1:500).

    Techniques: Expressing, Staining, Flow Cytometry, Two Tailed Test

    Viability staining of MSCs in HA hydrogel constructs after 28 days of in vitro culture for groups containing microspheres with TGF-β3 (MS+T), containing empty microspheres either in chondrogenic media (MS-T+CM) or in standard growth media (MS-T-CM), or with TGFβ-3 alone (T only). Green: live cells; Red: dead cells; scale bar = 100 μm.

    Journal: Biomaterials

    Article Title: Enhanced MSC Chondrogenesis Following Delivery of TGF-?3 from Alginate Microspheres within Hyaluronic Acid Hydrogels In Vitro and In Vivo

    doi: 10.1016/j.biomaterials.2011.05.033

    Figure Lengend Snippet: Viability staining of MSCs in HA hydrogel constructs after 28 days of in vitro culture for groups containing microspheres with TGF-β3 (MS+T), containing empty microspheres either in chondrogenic media (MS-T+CM) or in standard growth media (MS-T-CM), or with TGFβ-3 alone (T only). Green: live cells; Red: dead cells; scale bar = 100 μm.

    Article Snippet: Cell viability was assessed using the LIVE/DEAD Assay Kit (Molecular Probes) where live cells are stained green with calcein- AM and dead cells stained red with ethidium homodimer.

    Techniques: Staining, Construct, In Vitro, Mass Spectrometry

    Lipid electrophiles and PC protect against endothelial death and disintegrity dependent on Nrf2. MBMECs were subjected to lethal OGD after PC or 4-HNE treatment. (A) Live/Dead staining with (B) cell counting and (C) the LDH assay demonstrated increased cell survival and decreased cell death by both PC and 4-HNE. (D) In vitro BBB models were created by plating either MBMECs on the luminal side, or MBMECs (luminal side) and astrocytes (abluminal side) on each side. FITC-dextran was added to the luminal compartments, and dextran leakage was evaluated by measuring fluorescent intensities in the abluminal compartments 8 h after OGD. (E) OGD significantly increased dextran leakage in MBMEC cultures, which was partially reversed by both PC and 4-HNE. (F) In MBMEC-astrocyte cocultures, both PC and 4-HNE attenuated OGD-induced cell death, as indicated by the LDH assay, and (G) prevented dextran leakage. MBMECs were transfected by shRNA targeting a scrambled sequence (Sc) or Nrf2. (H) Live/Dead staining with (I) cell counting and (J) LDH assay demonstrated that PC- or 4-HNE-mediated protection was abolished by Nrf2 knockdown. ** and *** p

    Journal: Redox Biology

    Article Title: Brain ischemic preconditioning protects against ischemic injury and preserves the blood-brain barrier via oxidative signaling and Nrf2 activation

    doi: 10.1016/j.redox.2018.05.001

    Figure Lengend Snippet: Lipid electrophiles and PC protect against endothelial death and disintegrity dependent on Nrf2. MBMECs were subjected to lethal OGD after PC or 4-HNE treatment. (A) Live/Dead staining with (B) cell counting and (C) the LDH assay demonstrated increased cell survival and decreased cell death by both PC and 4-HNE. (D) In vitro BBB models were created by plating either MBMECs on the luminal side, or MBMECs (luminal side) and astrocytes (abluminal side) on each side. FITC-dextran was added to the luminal compartments, and dextran leakage was evaluated by measuring fluorescent intensities in the abluminal compartments 8 h after OGD. (E) OGD significantly increased dextran leakage in MBMEC cultures, which was partially reversed by both PC and 4-HNE. (F) In MBMEC-astrocyte cocultures, both PC and 4-HNE attenuated OGD-induced cell death, as indicated by the LDH assay, and (G) prevented dextran leakage. MBMECs were transfected by shRNA targeting a scrambled sequence (Sc) or Nrf2. (H) Live/Dead staining with (I) cell counting and (J) LDH assay demonstrated that PC- or 4-HNE-mediated protection was abolished by Nrf2 knockdown. ** and *** p

    Article Snippet: The Live/Dead cell viability assay was performed as previously described , according to the manufacturer's instructions (Molecular Probes, Eugene, OR).

    Techniques: Staining, Cell Counting, Lactate Dehydrogenase Assay, In Vitro, Transfection, shRNA, Sequencing

    Cell viability of encapsulated MSCs in the IPN hydrogel. (a–d) A fluorescent Live/Dead staining for the MSC-encapsulated hybrids treated with free swelling (FS), compressive loading at magnitude of 5% (5%), compressive loading at magnitude of 10% (10%), and compressive loading at magnitude of 20% (20%) (magnification: 40x). (e) Quantification of NPC percent (Live/Dead) for the fluorescent Live/Dead staining. (f) Measurement of the DNA content of MSC-seeded hybrids treated with free swelling and compressive loading at magnitudes of 5%, 10%, and 20%. Data were expressed as means ± SD ( n = 3). ∗ p

    Journal: Stem Cells International

    Article Title: Low Magnitude of Compression Enhances Biosynthesis of Mesenchymal Stem Cells towards Nucleus Pulposus Cells via the TRPV4-Dependent Pathway

    doi: 10.1155/2018/7061898

    Figure Lengend Snippet: Cell viability of encapsulated MSCs in the IPN hydrogel. (a–d) A fluorescent Live/Dead staining for the MSC-encapsulated hybrids treated with free swelling (FS), compressive loading at magnitude of 5% (5%), compressive loading at magnitude of 10% (10%), and compressive loading at magnitude of 20% (20%) (magnification: 40x). (e) Quantification of NPC percent (Live/Dead) for the fluorescent Live/Dead staining. (f) Measurement of the DNA content of MSC-seeded hybrids treated with free swelling and compressive loading at magnitudes of 5%, 10%, and 20%. Data were expressed as means ± SD ( n = 3). ∗ p

    Article Snippet: Live/Dead Assay The cell viability with different magnitudes of compression was investigated with the LIVE/DEAD Viability Assay Kit (Invitrogen, USA), according to the manufacturer's instructions.

    Techniques: Staining

    Assessment of murine islet viability and function 24 hrs following coating assembly. Coated islets were treated with NHS-PEG-N 3 /4armPEG-MDT/Alg-N 3 , while control islets were exposed to washing steps but not the polymers. Representative live/dead multi-slice projection confocal microscopy images of control (A) and coated (B) islets; (green=viable; red=dead). Metabolic activity, per MTT metabolic assay, (C) and glucose stimulated insulin secretion (GSIS) (D) of murine islets indicate high cytocompatibility of coating. *P

    Journal: Advanced healthcare materials

    Article Title: Long-term Survival of Allograft Murine Islets Coated via Covalently Stabilized Polymers

    doi: 10.1002/adhm.201300573

    Figure Lengend Snippet: Assessment of murine islet viability and function 24 hrs following coating assembly. Coated islets were treated with NHS-PEG-N 3 /4armPEG-MDT/Alg-N 3 , while control islets were exposed to washing steps but not the polymers. Representative live/dead multi-slice projection confocal microscopy images of control (A) and coated (B) islets; (green=viable; red=dead). Metabolic activity, per MTT metabolic assay, (C) and glucose stimulated insulin secretion (GSIS) (D) of murine islets indicate high cytocompatibility of coating. *P

    Article Snippet: Cell viability was visualized via LIVE/DEAD Viability/Cytotoxicity Assay Kit (Invitrogen) and imaged through a Leica SP5 Inverted Confocal Microscope.

    Techniques: Confocal Microscopy, Activity Assay, MTT Assay, Metabolic Assay

    Fluorescent LIVE/DEAD images of BAECs after exposure to hydrostatic pressure for either 2 days or 4 days. Cells were depressurized by either rapid depressurization (A) or slow depressurization (B) or exposed only to ambient atmospheric pressure (negative control) (C) and then stained as in Fig 2 . The vast majority of observed cells were live as indicated by the green stain. Scale Bar = 100 μm.

    Journal: PLoS ONE

    Article Title: The effect of the rate of hydrostatic pressure depressurization on cells in culture

    doi: 10.1371/journal.pone.0189890

    Figure Lengend Snippet: Fluorescent LIVE/DEAD images of BAECs after exposure to hydrostatic pressure for either 2 days or 4 days. Cells were depressurized by either rapid depressurization (A) or slow depressurization (B) or exposed only to ambient atmospheric pressure (negative control) (C) and then stained as in Fig 2 . The vast majority of observed cells were live as indicated by the green stain. Scale Bar = 100 μm.

    Article Snippet: Cell viability assays Following a 3 hour, 24 hour, 2 day, or 4 day application of elevated hydrostatic pressure, BAEC viability was visualized using a LIVE/DEAD viability kit for mammalian cells (Life Technologies, L3224).

    Techniques: Negative Control, Staining

    Fluorescent LIVE/DEAD images of BAECs after exposure to elevated hydrostatic pressure for 3 hours. Cells were depressurized by either rapid depressurization (A) or slow depressurization (B), or exposed only to ambient atmospheric pressure (negative control) (C), and then stained with LIVE/DEAD assay. Live cells are green; dead cells are stained red and indicated by the white arrows. Images represent data gathered from three replicates per experimental group. Each well was surveyed in its entirety and at least two representative images were taken for each experimental group. Scale Bar = 100 μm.

    Journal: PLoS ONE

    Article Title: The effect of the rate of hydrostatic pressure depressurization on cells in culture

    doi: 10.1371/journal.pone.0189890

    Figure Lengend Snippet: Fluorescent LIVE/DEAD images of BAECs after exposure to elevated hydrostatic pressure for 3 hours. Cells were depressurized by either rapid depressurization (A) or slow depressurization (B), or exposed only to ambient atmospheric pressure (negative control) (C), and then stained with LIVE/DEAD assay. Live cells are green; dead cells are stained red and indicated by the white arrows. Images represent data gathered from three replicates per experimental group. Each well was surveyed in its entirety and at least two representative images were taken for each experimental group. Scale Bar = 100 μm.

    Article Snippet: Cell viability assays Following a 3 hour, 24 hour, 2 day, or 4 day application of elevated hydrostatic pressure, BAEC viability was visualized using a LIVE/DEAD viability kit for mammalian cells (Life Technologies, L3224).

    Techniques: Negative Control, Staining, Live Dead Assay

    The results of the LIVE/DEAD ® Viability/Cytotoxicity assay of adipose tissue-derived stem cells (ADSCs). The ADSCs cultured in the two-dimensional (2D) plate ( A ), and in three-dimensional (3D) hydrogel ( B ) (Scale bars, 100 μm). The quantitative results of the LIVE/DEAD ® Viability/Cytotoxicity assay ( C ). Quantitative analysis of the ADSC proliferation rate using the cell counting kit-8 (CCK-8) assay during 7 days ( D ).

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Three-Dimensional Printed Titanium Scaffolds Enhance Osteogenic Differentiation and New Bone Formation by Cultured Adipose Tissue-Derived Stem Cells Through the IGF-1R/AKT/Mammalian Target of Rapamycin Complex 1 (mTORC1) Pathway

    doi: 10.12659/MSM.918517

    Figure Lengend Snippet: The results of the LIVE/DEAD ® Viability/Cytotoxicity assay of adipose tissue-derived stem cells (ADSCs). The ADSCs cultured in the two-dimensional (2D) plate ( A ), and in three-dimensional (3D) hydrogel ( B ) (Scale bars, 100 μm). The quantitative results of the LIVE/DEAD ® Viability/Cytotoxicity assay ( C ). Quantitative analysis of the ADSC proliferation rate using the cell counting kit-8 (CCK-8) assay during 7 days ( D ).

    Article Snippet: Cell viability and proliferation After 7 days of culture in the 2D plates or 3D hydrogel, the cells were evaluated with the LIVE/DEAD® Viability/Cytotoxicity assay kit (L3224) (Invitrogen, Carlsbad, CA, USA).

    Techniques: Cytotoxicity Assay, Derivative Assay, Cell Culture, Cell Counting, CCK-8 Assay

    Interferon regulatory factor 5 (IRF5) is required for toll-like receptor 9/B cell receptor -induced antibody secreting cell differentiation. Isolated human naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and mock-stimulated or stimulated with the indicated cocktails. (A) IRF5 transcript expression was quantified through qPCR on RNA isolated 48 h postnucleofection and 12 h poststimulation with anti-IgM + CpG-B (two-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (B) Protein lysates were prepared from nucleofected B cells 72 h postnucleofection and 24 h poststimulation with anti-IgM + CpG-B. Western blot is one representative experiment out of three performed on n = 3 independent donors. (C) Representative histograms of IRF5 expression 48 h postnucleofection. Viable cells were analyzed through live/dead staining discrimination. (D) Plotted MFI of IRF5 from (C) (one-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (E) Plotted percentage of B cell viability assessed 72 h post-nucleofection, as determined through trypan blue exclusion. Data are from n = 3 independent donors. (F) Representative dot plots from B cell apoptosis quantified following staining with Annexin V and 7 amino-actinomycin D (7-AAD). Early apoptotic events are characterized as Annexin V + 7AAD − , whereas late apoptotic events are Annexin V + 7AAD + . Quantitation is shown in (G) . (G) Average apoptotic B cells from (F) 96 h post-nucleofection and 48 h post-stimulation (Two-way ANOVA with Tukey’s post hoc test; n = 3 independent donors). (H) Isolated naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and stimulated with either CD40L or the combination of CD40L, IL21, anti-IgM, and CpG-B for 7 days. Plasmablast differentiation was quantified through gating of CD19 + CD20 + IgD − CD27 + CD38 + B cells; final CD27 + CD38 + gating is shown. Flow cytometry contour plots are representative of one experiment from a single donor. (I) Average number of plasmablasts from (H) following culture for 7 days (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). (J) Average number of IgD + CD38 lo B cells from (H) following stimulation (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). Error bars represent SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Journal: Frontiers in Immunology

    Article Title: B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation

    doi: 10.3389/fimmu.2017.01938

    Figure Lengend Snippet: Interferon regulatory factor 5 (IRF5) is required for toll-like receptor 9/B cell receptor -induced antibody secreting cell differentiation. Isolated human naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and mock-stimulated or stimulated with the indicated cocktails. (A) IRF5 transcript expression was quantified through qPCR on RNA isolated 48 h postnucleofection and 12 h poststimulation with anti-IgM + CpG-B (two-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (B) Protein lysates were prepared from nucleofected B cells 72 h postnucleofection and 24 h poststimulation with anti-IgM + CpG-B. Western blot is one representative experiment out of three performed on n = 3 independent donors. (C) Representative histograms of IRF5 expression 48 h postnucleofection. Viable cells were analyzed through live/dead staining discrimination. (D) Plotted MFI of IRF5 from (C) (one-way ANOVA with Tukey’s post hoc test; n = 4 independent donors). (E) Plotted percentage of B cell viability assessed 72 h post-nucleofection, as determined through trypan blue exclusion. Data are from n = 3 independent donors. (F) Representative dot plots from B cell apoptosis quantified following staining with Annexin V and 7 amino-actinomycin D (7-AAD). Early apoptotic events are characterized as Annexin V + 7AAD − , whereas late apoptotic events are Annexin V + 7AAD + . Quantitation is shown in (G) . (G) Average apoptotic B cells from (F) 96 h post-nucleofection and 48 h post-stimulation (Two-way ANOVA with Tukey’s post hoc test; n = 3 independent donors). (H) Isolated naive B cells were nucleofected with 500 nM of mock, scrambled or IRF5 siRNA and stimulated with either CD40L or the combination of CD40L, IL21, anti-IgM, and CpG-B for 7 days. Plasmablast differentiation was quantified through gating of CD19 + CD20 + IgD − CD27 + CD38 + B cells; final CD27 + CD38 + gating is shown. Flow cytometry contour plots are representative of one experiment from a single donor. (I) Average number of plasmablasts from (H) following culture for 7 days (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). (J) Average number of IgD + CD38 lo B cells from (H) following stimulation (two-way ANOVA with Tukey’s post hoc test; n = 9 independent donors). Error bars represent SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

    Article Snippet: Flow Cytometry Analysis Isolated B cells were washed and stained with Live/Dead viability discrimination dye (Life Tech #L34968).

    Techniques: Cell Differentiation, Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Staining, Quantitation Assay, Flow Cytometry, Cytometry

    The sperm viability, as assessed by a fluorescent staining method. Live sperm cells fluoresced green (SYBR-14 dye), and dead sperm cells fluoresced red (PI) (1600×). (A) Control live sperm appeared green, confirming the utility of the assay. The spermatozoa treated with 0.20 mM N-9 (B) and 0.20 mM PD (C) both exhibited PI staining (red). (D) The sperm count showed that 0.20 mM PD killed almost all of the sperm, while 0.20 mM of N-9 only killed 65.5% of the spermatozoa. Each bar represents the mean ± SEM of six sets of observations; *: Significantly different from the control, p

    Journal: PLoS ONE

    Article Title: Evaluation of the Spermicidal and Contraceptive Activity of Platycodin D, a Saponin from Platycodon grandiflorum

    doi: 10.1371/journal.pone.0082068

    Figure Lengend Snippet: The sperm viability, as assessed by a fluorescent staining method. Live sperm cells fluoresced green (SYBR-14 dye), and dead sperm cells fluoresced red (PI) (1600×). (A) Control live sperm appeared green, confirming the utility of the assay. The spermatozoa treated with 0.20 mM N-9 (B) and 0.20 mM PD (C) both exhibited PI staining (red). (D) The sperm count showed that 0.20 mM PD killed almost all of the sperm, while 0.20 mM of N-9 only killed 65.5% of the spermatozoa. Each bar represents the mean ± SEM of six sets of observations; *: Significantly different from the control, p

    Article Snippet: The LIVE/DEAD sperm viability kit (cat. L-7011) was obtained from Invitrogen (Molecular Probes Inc., Eugene, OR, USA).

    Techniques: Staining

    Spermatozoa with loosely attached particles after the washing steps of the fluorescent labelling with LIVE/DEAD Fixable red probe. Confocal laser scanning microscopy

    Journal: Acta Veterinaria Scandinavica

    Article Title: Ejaculated boar spermatozoa displaying a rare multivesicular defect

    doi: 10.1186/s13028-018-0375-7

    Figure Lengend Snippet: Spermatozoa with loosely attached particles after the washing steps of the fluorescent labelling with LIVE/DEAD Fixable red probe. Confocal laser scanning microscopy

    Article Snippet: Spermatozoa were labelled with LIVE/DEAD® Fixable Red Dead Cell Stain Kit (L23102, Invitrogen).

    Techniques: Confocal Laser Scanning Microscopy

    HBD3-C15 and Nystatin (Nys) showed fungicidal activity against C. albicans . C. albicans (6 × 10 6 cells/mL) was incubated for 48 hr on cover glass and treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, 300 µg/mL), aqueous calcium hydroxide (CH, 100 µg/mL), and Nys (20 µg/mL) for 7 days. (a) FilmTracer LIVE/DEAD Biofilm Viability staining was performed and observed under CLSM; (b) The numbers of dead cells (PI stained red) were increased in the group of HBD3-C15 in a dose-dependent manner; (c) The dead cell biomass were significantly higher in the groups of HBD3-C15 (≥ 100 µg/mL) and Nys than CH group ( p

    Journal: Restorative Dentistry & Endodontics

    Article Title: Antifungal effects of synthetic human β-defensin 3-C15 peptide

    doi: 10.5395/rde.2016.41.2.91

    Figure Lengend Snippet: HBD3-C15 and Nystatin (Nys) showed fungicidal activity against C. albicans . C. albicans (6 × 10 6 cells/mL) was incubated for 48 hr on cover glass and treated with either HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, 300 µg/mL), aqueous calcium hydroxide (CH, 100 µg/mL), and Nys (20 µg/mL) for 7 days. (a) FilmTracer LIVE/DEAD Biofilm Viability staining was performed and observed under CLSM; (b) The numbers of dead cells (PI stained red) were increased in the group of HBD3-C15 in a dose-dependent manner; (c) The dead cell biomass were significantly higher in the groups of HBD3-C15 (≥ 100 µg/mL) and Nys than CH group ( p

    Article Snippet: Finally they were stained with the FilmTracer LIVE/DEAD Biofilm viability kit (Molecular Probes, Carlsbad, CA, USA), which uses SYTO9 and propidium iodide (PI) to stain live and dead cells within biofilms respectively.

    Techniques: Activity Assay, Incubation, Staining, Confocal Laser Scanning Microscopy

    Phloxine B staining reflects percentage of dead cells. (A) Example of colony redness score extraction by pyphe-quantify in redness mode. From the acquired input image (i), colors are enhanced and the background subtracted (ii), colonies are identified by local thresholding (iii), and redness is quantified and annotated in the original image (iv). (B) Representative cells for alive, dead and lysed cells using imaging flow cytometry (ImageStream) analysis. Lysed cells show no signal in either the phloxine B or LIVE/DEAD channels. Live cells show an intermediate signal intensity in the phloxine B channel but no LIVE/DEAD signal. Dead cells are brightly stained in both channels. (C) Histogram of intensities in phloxine B channel across 23 samples with three populations (lysed, alive and dead) clearly resolved. (D) Fraction of live cells (live/(lysed+dead)) by ImageStream correlate with colony redness scores (corrected by row/median column normalisation) obtained with pyphe. (E) Co-localisation of phloxine B stain with LIVE/DEAD stain for the standard lab strain 972 . (F) Comparison of Phloxine B staining with LIVE/DEAD stain by ImageStream. Both readouts agree with 99.3% accuracy using the illustrated thresholds.

    Journal: bioRxiv

    Article Title: Pyphe: A python toolbox for assessing microbial growth and cell viability in high-throughput colony screens

    doi: 10.1101/2020.01.22.915363

    Figure Lengend Snippet: Phloxine B staining reflects percentage of dead cells. (A) Example of colony redness score extraction by pyphe-quantify in redness mode. From the acquired input image (i), colors are enhanced and the background subtracted (ii), colonies are identified by local thresholding (iii), and redness is quantified and annotated in the original image (iv). (B) Representative cells for alive, dead and lysed cells using imaging flow cytometry (ImageStream) analysis. Lysed cells show no signal in either the phloxine B or LIVE/DEAD channels. Live cells show an intermediate signal intensity in the phloxine B channel but no LIVE/DEAD signal. Dead cells are brightly stained in both channels. (C) Histogram of intensities in phloxine B channel across 23 samples with three populations (lysed, alive and dead) clearly resolved. (D) Fraction of live cells (live/(lysed+dead)) by ImageStream correlate with colony redness scores (corrected by row/median column normalisation) obtained with pyphe. (E) Co-localisation of phloxine B stain with LIVE/DEAD stain for the standard lab strain 972 . (F) Comparison of Phloxine B staining with LIVE/DEAD stain by ImageStream. Both readouts agree with 99.3% accuracy using the illustrated thresholds.

    Article Snippet: For analysis of phloxine B and LIVE/DEAD co-staining, 500µL of the same suspension were centrifuged at 4000g for 2 min, the supernatant was removed and the pellet resuspended in 300µL of LIVE/DEAD solution (LIVE/DEAD™ Fixable Far Red Dead Cell Stain Kit, for 633 or 635 nm excitation, ThermoFisher Scientific, Cat. no. L34974).

    Techniques: Staining, Imaging, Flow Cytometry