permeabilization  (Thermo Fisher)


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    Image iT Fixation Permeabilization Kit
    Description:
    The Image iT Fixation Permeabilization Kit is a convenient to use fixation permeabilization kit that contains all the necessary reagents to prepare your cells for antibody staining and imaging The high quality components help preserve cell morphology and reduce background staining and come in a practical box with single use vials and easy to follow protocols Note the 4 formadehyde methanol free fixative solution in this kit is also available for separate purchase see Cat No R37814 • A complete fixation permeabilization kit with ready to use components and room temperature storage • Optimized for secondary antibody labeling fixed cell dye staining and GFP applications • Reduced background staining See other ReadyProbes reagents for cell staining Find more tools for fixed cell imaging The Image iT Fixation Permeabilization Kit Contains • Fixative High purity 4 formaldehyde in PBS pH 7 3 • Permeabilization solution 0 5 Triton X 100 • Blocking buffer 3 BSA fraction V de lipidated New Zealand source in DPBS • Wash solution PBS pH 7 4
    Catalog Number:
    r37602
    Price:
    None
    Applications:
    Cell Analysis|Cellular Imaging|IHC Staining & Detection|Immunocytochemistry (ICC)|Immunofluorescence (IF)|Immunofluorescence Sample Preparation|Immunohistochemistry (IHC)
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher permeabilization
    E-Syt3ΔC2C redistribution in transfected adipocytes through the early steps of LD assembly and growth. Growth of E-Syt3ΔC2C coated lipid droplets by homotypic fusion. Live cell time lapse fluorescence/bright field microscopy of adipocytes transfected with EGFP-E-Syt3ΔC2C on day 4 of differentiation. Selected paired (A, B) and single (C) images from videos 3 and 4 show the encapsulation of newborn LDs within cisterna like structures containing the truncated fluorescent protein laid over the birefringent capsule (A, arrows); due to the loss of small amounts of NLs during the cell <t>permeabilization/fixation</t> it is uncertain to asses if the absence of NL in some cisternae was real (see Fig 3F ). Note the ongoing fusion between the cisternae (B, arrows), as well as between the encapsulated LDs (C, red arrows), and the predation of small LDs by the large LDs (C, white arrows). Scale bars = 2.65 μm (A), 1.97 μm (B), or 3.76 μm (C). (D-G) The dynamics of LD fusion in control adipocytes stained with LipidTOX (D, video 6) and in adipocytes transfected with EGFP E-Syt3ΔC2C (E-G, video 5). Snapshots show the LDs at the indicated recording times (right bottom). Line graphs represent the fusion profiles. Observe the fluorescent rings produced by the incorporation of EGFP E-Syt3ΔC2C into the surface of the LDs (E 1-2 ; F 1-2 ; G 1-2 ; Video 6). Fusion events were quantified by measuring the changes in the number, average size, and total area in two separate populations of small (
    The Image iT Fixation Permeabilization Kit is a convenient to use fixation permeabilization kit that contains all the necessary reagents to prepare your cells for antibody staining and imaging The high quality components help preserve cell morphology and reduce background staining and come in a practical box with single use vials and easy to follow protocols Note the 4 formadehyde methanol free fixative solution in this kit is also available for separate purchase see Cat No R37814 • A complete fixation permeabilization kit with ready to use components and room temperature storage • Optimized for secondary antibody labeling fixed cell dye staining and GFP applications • Reduced background staining See other ReadyProbes reagents for cell staining Find more tools for fixed cell imaging The Image iT Fixation Permeabilization Kit Contains • Fixative High purity 4 formaldehyde in PBS pH 7 3 • Permeabilization solution 0 5 Triton X 100 • Blocking buffer 3 BSA fraction V de lipidated New Zealand source in DPBS • Wash solution PBS pH 7 4
    https://www.bioz.com/result/permeabilization/product/Thermo Fisher
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    Images

    1) Product Images from "The Step-Wise C-Truncation and Transport of ESyt3 to Lipid Droplets Reveals a Mother Primordial Cisterna"

    Article Title: The Step-Wise C-Truncation and Transport of ESyt3 to Lipid Droplets Reveals a Mother Primordial Cisterna

    Journal: bioRxiv

    doi: 10.1101/2020.07.17.209122

    E-Syt3ΔC2C redistribution in transfected adipocytes through the early steps of LD assembly and growth. Growth of E-Syt3ΔC2C coated lipid droplets by homotypic fusion. Live cell time lapse fluorescence/bright field microscopy of adipocytes transfected with EGFP-E-Syt3ΔC2C on day 4 of differentiation. Selected paired (A, B) and single (C) images from videos 3 and 4 show the encapsulation of newborn LDs within cisterna like structures containing the truncated fluorescent protein laid over the birefringent capsule (A, arrows); due to the loss of small amounts of NLs during the cell permeabilization/fixation it is uncertain to asses if the absence of NL in some cisternae was real (see Fig 3F ). Note the ongoing fusion between the cisternae (B, arrows), as well as between the encapsulated LDs (C, red arrows), and the predation of small LDs by the large LDs (C, white arrows). Scale bars = 2.65 μm (A), 1.97 μm (B), or 3.76 μm (C). (D-G) The dynamics of LD fusion in control adipocytes stained with LipidTOX (D, video 6) and in adipocytes transfected with EGFP E-Syt3ΔC2C (E-G, video 5). Snapshots show the LDs at the indicated recording times (right bottom). Line graphs represent the fusion profiles. Observe the fluorescent rings produced by the incorporation of EGFP E-Syt3ΔC2C into the surface of the LDs (E 1-2 ; F 1-2 ; G 1-2 ; Video 6). Fusion events were quantified by measuring the changes in the number, average size, and total area in two separate populations of small (
    Figure Legend Snippet: E-Syt3ΔC2C redistribution in transfected adipocytes through the early steps of LD assembly and growth. Growth of E-Syt3ΔC2C coated lipid droplets by homotypic fusion. Live cell time lapse fluorescence/bright field microscopy of adipocytes transfected with EGFP-E-Syt3ΔC2C on day 4 of differentiation. Selected paired (A, B) and single (C) images from videos 3 and 4 show the encapsulation of newborn LDs within cisterna like structures containing the truncated fluorescent protein laid over the birefringent capsule (A, arrows); due to the loss of small amounts of NLs during the cell permeabilization/fixation it is uncertain to asses if the absence of NL in some cisternae was real (see Fig 3F ). Note the ongoing fusion between the cisternae (B, arrows), as well as between the encapsulated LDs (C, red arrows), and the predation of small LDs by the large LDs (C, white arrows). Scale bars = 2.65 μm (A), 1.97 μm (B), or 3.76 μm (C). (D-G) The dynamics of LD fusion in control adipocytes stained with LipidTOX (D, video 6) and in adipocytes transfected with EGFP E-Syt3ΔC2C (E-G, video 5). Snapshots show the LDs at the indicated recording times (right bottom). Line graphs represent the fusion profiles. Observe the fluorescent rings produced by the incorporation of EGFP E-Syt3ΔC2C into the surface of the LDs (E 1-2 ; F 1-2 ; G 1-2 ; Video 6). Fusion events were quantified by measuring the changes in the number, average size, and total area in two separate populations of small (

    Techniques Used: Transfection, Fluorescence, Microscopy, Staining, Produced

    2) Product Images from "The Extracellular Matrix Influences Ovarian Carcinoma Cells’ Sensitivity to Cisplatinum: A First Step towards Personalized Medicine"

    Article Title: The Extracellular Matrix Influences Ovarian Carcinoma Cells’ Sensitivity to Cisplatinum: A First Step towards Personalized Medicine

    Journal: Cancers

    doi: 10.3390/cancers12051175

    Ovarian cancer adherent cells and spheroids were characterized for the expression of different markers. We evaluated the expression of several markers for ovarian cancer cells by immunofluorescence, upon fixation and permeabilization. Adherent cells ( A ) appeared positively stained for vimentin, cytokeratin 8/18, mucin 1, CD44 and WT-1 and negatively stained for von Willebrand factor and CD45, excluding endothelial cell and blood cell contamination. Spheroids ( B ), after cytocentrifugation and fixation, were stained for vimentin, cytokeratin 8/18, mucin 1, CD44 and WT-1. Nuclei were stained with DAPI. Original magnification: 100×.
    Figure Legend Snippet: Ovarian cancer adherent cells and spheroids were characterized for the expression of different markers. We evaluated the expression of several markers for ovarian cancer cells by immunofluorescence, upon fixation and permeabilization. Adherent cells ( A ) appeared positively stained for vimentin, cytokeratin 8/18, mucin 1, CD44 and WT-1 and negatively stained for von Willebrand factor and CD45, excluding endothelial cell and blood cell contamination. Spheroids ( B ), after cytocentrifugation and fixation, were stained for vimentin, cytokeratin 8/18, mucin 1, CD44 and WT-1. Nuclei were stained with DAPI. Original magnification: 100×.

    Techniques Used: Expressing, Immunofluorescence, Staining

    3) Product Images from "Novel strategy for treating neurotropic viral infections using hypolipidemic drug Atorvastatin"

    Article Title: Novel strategy for treating neurotropic viral infections using hypolipidemic drug Atorvastatin

    Journal: bioRxiv

    doi: 10.1101/639096

    Reduction in hnRNPC abundance upon treatment with AT in virus-infected brain. (A and B) Balb C mice were infected with CHPV and JEV. AT treatment was administered once daily. Following appearance of signs of virus infection, brain samples were collected followed by fixation, generation of brain sections, permeabilization and stained with antibodies for detection of viral proteins and hnRNPC Scale bar 50 µm. Data presented are representative of three independent experiments.
    Figure Legend Snippet: Reduction in hnRNPC abundance upon treatment with AT in virus-infected brain. (A and B) Balb C mice were infected with CHPV and JEV. AT treatment was administered once daily. Following appearance of signs of virus infection, brain samples were collected followed by fixation, generation of brain sections, permeabilization and stained with antibodies for detection of viral proteins and hnRNPC Scale bar 50 µm. Data presented are representative of three independent experiments.

    Techniques Used: Infection, Mouse Assay, Staining

    4) Product Images from "Toxoplasma gondii inactivates human plasmacytoid dendritic cells by functional mimicry of IL-10"

    Article Title: Toxoplasma gondii inactivates human plasmacytoid dendritic cells by functional mimicry of IL-10

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

    doi: 10.4049/jimmunol.1701045

    T. gondii inhibits nuclear translocation of IRF-7 in response to HSV-1 without affecting phosphorylation of IRF-7 A , PBMC were pre-infected with T. gondii at an MOI of 4, followed by stimulation with HSV, MOI 1. 3hrs after stimulation with virus, fixed pDC were stained using anti-HLA-DR and CD123; following ethanol permeabilization intracellular phosphorylation of IRF7 was assessed using pIRF7-PE (pS477/pS479) and measured using flow cytometry. Representative flow plots from a single experiment are shown in the top panel, and summary data are shown in the lower panel for 3 independent experiments. B , Enriched pDC were pre-infected with T. gondii as described above and exposed to HSV-1 for 2, 4 and 6 hrs; BDCA2 and BDCA4 positive pDC were then analyzed for cytoplasmic and nuclear distribution of IRF7 using ImageStream and Amnis IDEAS software. Representative data are shown from the 4-hr timepoint. IRF7 nuclear translocation in pDC was determined by first creating an object mask for nuclear stain (DRAQ5), and then analyzed using built in function of similarity between IRF7 and nuclear mask. A histogram overlay was generated for comparison of IRF7 nuclear translocation in Mock (gray), HSV-1 (red), and in T. gondii pre-infected and HSV-1 treated pDC (green) pDC. Arrows indicate bin with representative images of not-translocated (left) and translocated (right) events. C , Kinetics of IRF7 nuclear translocation and the effect of T. gondii infection on IRF7 translocation in B were analyzed for statistical significance (n=3).
    Figure Legend Snippet: T. gondii inhibits nuclear translocation of IRF-7 in response to HSV-1 without affecting phosphorylation of IRF-7 A , PBMC were pre-infected with T. gondii at an MOI of 4, followed by stimulation with HSV, MOI 1. 3hrs after stimulation with virus, fixed pDC were stained using anti-HLA-DR and CD123; following ethanol permeabilization intracellular phosphorylation of IRF7 was assessed using pIRF7-PE (pS477/pS479) and measured using flow cytometry. Representative flow plots from a single experiment are shown in the top panel, and summary data are shown in the lower panel for 3 independent experiments. B , Enriched pDC were pre-infected with T. gondii as described above and exposed to HSV-1 for 2, 4 and 6 hrs; BDCA2 and BDCA4 positive pDC were then analyzed for cytoplasmic and nuclear distribution of IRF7 using ImageStream and Amnis IDEAS software. Representative data are shown from the 4-hr timepoint. IRF7 nuclear translocation in pDC was determined by first creating an object mask for nuclear stain (DRAQ5), and then analyzed using built in function of similarity between IRF7 and nuclear mask. A histogram overlay was generated for comparison of IRF7 nuclear translocation in Mock (gray), HSV-1 (red), and in T. gondii pre-infected and HSV-1 treated pDC (green) pDC. Arrows indicate bin with representative images of not-translocated (left) and translocated (right) events. C , Kinetics of IRF7 nuclear translocation and the effect of T. gondii infection on IRF7 translocation in B were analyzed for statistical significance (n=3).

    Techniques Used: Translocation Assay, Infection, Staining, Flow Cytometry, Software, Generated

    5) Product Images from "Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage"

    Article Title: Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.180810

    DIC-based corrections of image displacements during the full on-stage ICC. Images were acquired using a CCD camera with high spatial resolution but slow acquisition. Cultured mouse hippocampal neurons were imaged with DIC optics. All panels represent overlays of two images. The DIC image acquired during live-cell imaging (image #1) was used as a spatial reference image and was pseudo-colored red in all panels. DIC images were further acquired during later procedures: immediately after completing chemical fixation (image #2, A ), immediately after membrane permeabilization (image #3, B ), and after completion of ICC procedures (image #4, C ). These test images were pseudo-colored green. In A, B, C , top and bottom rows represent overlays before and after correcting the displacements, respectively. Displacements between two images appear as red or green, and no displacements appear yellow. The degree of displacement, and therefore the degree of required correction, is indicated by how much the test image had to be translated laterally along the x- and y-axes to match the reference image [(Δx, Δy) in pixels]. For a simple demonstration, the illustrated images were selected from the ones acquired without fluorescent filters.
    Figure Legend Snippet: DIC-based corrections of image displacements during the full on-stage ICC. Images were acquired using a CCD camera with high spatial resolution but slow acquisition. Cultured mouse hippocampal neurons were imaged with DIC optics. All panels represent overlays of two images. The DIC image acquired during live-cell imaging (image #1) was used as a spatial reference image and was pseudo-colored red in all panels. DIC images were further acquired during later procedures: immediately after completing chemical fixation (image #2, A ), immediately after membrane permeabilization (image #3, B ), and after completion of ICC procedures (image #4, C ). These test images were pseudo-colored green. In A, B, C , top and bottom rows represent overlays before and after correcting the displacements, respectively. Displacements between two images appear as red or green, and no displacements appear yellow. The degree of displacement, and therefore the degree of required correction, is indicated by how much the test image had to be translated laterally along the x- and y-axes to match the reference image [(Δx, Δy) in pixels]. For a simple demonstration, the illustrated images were selected from the ones acquired without fluorescent filters.

    Techniques Used: Immunocytochemistry, Cell Culture, Live Cell Imaging

    6) Product Images from "The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization"

    Article Title: The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00788-15

    Immunofluorescence analysis of internalization of CdtB CRAC site mutants in Jurkat cells. Jurkat cells were exposed to medium alone (gray curves), CdtA and CdtC alone (A), and CdtA and CdtC in the presence of CdtB WT (B), CdtB V104P (C), CdtB Y105P (D), CdtB Y107P (E), or CdtB R110P (F) for 1 h and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization, and staining with anti-CdtB MAb conjugated to Alexa Fluor 488. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF). Note that the MCF for cells not exposed to any Cdt peptide was 5.7. At least 10,000 cells were analyzed per sample; results are representative of three experiments.
    Figure Legend Snippet: Immunofluorescence analysis of internalization of CdtB CRAC site mutants in Jurkat cells. Jurkat cells were exposed to medium alone (gray curves), CdtA and CdtC alone (A), and CdtA and CdtC in the presence of CdtB WT (B), CdtB V104P (C), CdtB Y105P (D), CdtB Y107P (E), or CdtB R110P (F) for 1 h and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization, and staining with anti-CdtB MAb conjugated to Alexa Fluor 488. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF). Note that the MCF for cells not exposed to any Cdt peptide was 5.7. At least 10,000 cells were analyzed per sample; results are representative of three experiments.

    Techniques Used: Immunofluorescence, Flow Cytometry, Cytometry, Staining, Fluorescence

    Immunofluorescence analysis of internalization of CdtB CRAC site mutants in THP-1-derived macrophages. Macrophages were exposed to medium alone (gray curves), CdtA and CdtC alone (A), and CdtA and CdtC in the presence of CdtB WT (B), CdtB V104P (C), CdtB Y105P (D), CdtB Y107P (E), or CdtB R110P (F) for 1 h and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization, and staining with anti-CdtB MAb conjugated to Alexa Fluor 488. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF). Note that the MCF for cells not exposed to any Cdt peptide was 8.6. At least 10,000 cells were analyzed per sample; results are representative of three experiments.
    Figure Legend Snippet: Immunofluorescence analysis of internalization of CdtB CRAC site mutants in THP-1-derived macrophages. Macrophages were exposed to medium alone (gray curves), CdtA and CdtC alone (A), and CdtA and CdtC in the presence of CdtB WT (B), CdtB V104P (C), CdtB Y105P (D), CdtB Y107P (E), or CdtB R110P (F) for 1 h and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization, and staining with anti-CdtB MAb conjugated to Alexa Fluor 488. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF). Note that the MCF for cells not exposed to any Cdt peptide was 8.6. At least 10,000 cells were analyzed per sample; results are representative of three experiments.

    Techniques Used: Immunofluorescence, Derivative Assay, Flow Cytometry, Cytometry, Staining, Fluorescence

    Cumulative results of immunofluorescence assessment of CdtB internalization and Cdt binding. (A) Results obtained from three experiments for internalization of CdtB in Jurkat cells. (B) Results obtained from three experiments for internalization of CdtB in THP-1 cells. (C) Results obtained from three experiments for holotoxin binding to the cell surface determined by immunofluorescence staining for the presence of CdtC in the absence of permeabilization. Results in each panel are expressed as a percentage of the MCF observed in CdtB WT and represent the means ± SEM *, P
    Figure Legend Snippet: Cumulative results of immunofluorescence assessment of CdtB internalization and Cdt binding. (A) Results obtained from three experiments for internalization of CdtB in Jurkat cells. (B) Results obtained from three experiments for internalization of CdtB in THP-1 cells. (C) Results obtained from three experiments for holotoxin binding to the cell surface determined by immunofluorescence staining for the presence of CdtC in the absence of permeabilization. Results in each panel are expressed as a percentage of the MCF observed in CdtB WT and represent the means ± SEM *, P

    Techniques Used: Immunofluorescence, Binding Assay, Staining

    7) Product Images from "Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry"

    Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194593

    Effects of different buffers on the detection of rare populations. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). This data compare the frequency of various CD45+ populations between the CS condition and the different permeabilization conditions. a.) Frequencies of rare CD4+ T cell populations: primed T cells (CXCR5+ CCR7+) and Treg cells (CD25 hi CD127 low ). b.) Frequencies of rare B cell populations such as transitional B cells (CD24 hi CD38 hi ) and un-switched memory B cells (CD19+IgD+CD27+). c.) Statistics showing the comparison of the frequency of rare T and B cell populations within the different experimental conditions. The concentrations of antibodies used were: CXCR5 (0.04 mg/ml), CCR7 (0.5 mg/ml), CD25 (0.5 mg/ml), CD127 (0.5 mg/ml), CD24 (0.3 mg/ml), CD38 (0.3 mg/ml), IgD (0.25 mg/ml) and CD27 (0.1 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Effects of different buffers on the detection of rare populations. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). This data compare the frequency of various CD45+ populations between the CS condition and the different permeabilization conditions. a.) Frequencies of rare CD4+ T cell populations: primed T cells (CXCR5+ CCR7+) and Treg cells (CD25 hi CD127 low ). b.) Frequencies of rare B cell populations such as transitional B cells (CD24 hi CD38 hi ) and un-switched memory B cells (CD19+IgD+CD27+). c.) Statistics showing the comparison of the frequency of rare T and B cell populations within the different experimental conditions. The concentrations of antibodies used were: CXCR5 (0.04 mg/ml), CCR7 (0.5 mg/ml), CD25 (0.5 mg/ml), CD127 (0.5 mg/ml), CD24 (0.3 mg/ml), CD38 (0.3 mg/ml), IgD (0.25 mg/ml) and CD27 (0.1 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Staining

    Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Incubation, Staining

    Visualization of the effects of different buffers on the intensity of cell surface markers. Cells from healthy donors were labeled with antibodies targeting both cell surface antigens and intranuclear antigens. Data show the distribution of CD45+ live cells on viSNE plots with the “cell surface staining” only condition (CS) and the different permeabilization conditions: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). The intensity of CD19+, CD16+, CD56+, CD14+ and HLADR+ events are shown. The concentrations of antibodies used for the detection of these markers are as follows: CD19 (0.5 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). viSNE was performed using 1000 iterations, with a perplexity of 30 and theta = 0.3. The data shown are representative of an independent experiment and represent median with interquartile. Experiments were performed 3 times independently. Different healthy individuals were used for each independent experiment.
    Figure Legend Snippet: Visualization of the effects of different buffers on the intensity of cell surface markers. Cells from healthy donors were labeled with antibodies targeting both cell surface antigens and intranuclear antigens. Data show the distribution of CD45+ live cells on viSNE plots with the “cell surface staining” only condition (CS) and the different permeabilization conditions: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). The intensity of CD19+, CD16+, CD56+, CD14+ and HLADR+ events are shown. The concentrations of antibodies used for the detection of these markers are as follows: CD19 (0.5 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). viSNE was performed using 1000 iterations, with a perplexity of 30 and theta = 0.3. The data shown are representative of an independent experiment and represent median with interquartile. Experiments were performed 3 times independently. Different healthy individuals were used for each independent experiment.

    Techniques Used: Labeling, Staining

    Partial loss of the signal intensity of CD4 and CD127 after barcoding. Cells were thawed as described above and used for the following purposes: a) assessment of the effects of barcoding on the expression of surface markers. Three experimental conditions were performed: no barcode/no permeabilization, surface staining before barcoding and finally barcoding before surface staining. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The concentrations of antibodies used are as follow: CD45 (0.5 mg/ml), CD3 (0.08 mg/ml), CD4 (0.25 mg/ml), CD127 (0.5 mg/ml) and CD25 (0.5 mg/ml). b) Assessment of the cause of the lower signal intensity of CD4 and CD127 when PBMC are barcoded before cell surface staining. 3 conditions were evaluated: no barcode/no permeabilization, permeabilization only/no barcoding and permeabilization followed by barcoding. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The data shown are representative of an independent experiment. Experiments were performed 3 times independently. PBMC from different individuals were used for each independent experiment.
    Figure Legend Snippet: Partial loss of the signal intensity of CD4 and CD127 after barcoding. Cells were thawed as described above and used for the following purposes: a) assessment of the effects of barcoding on the expression of surface markers. Three experimental conditions were performed: no barcode/no permeabilization, surface staining before barcoding and finally barcoding before surface staining. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The concentrations of antibodies used are as follow: CD45 (0.5 mg/ml), CD3 (0.08 mg/ml), CD4 (0.25 mg/ml), CD127 (0.5 mg/ml) and CD25 (0.5 mg/ml). b) Assessment of the cause of the lower signal intensity of CD4 and CD127 when PBMC are barcoded before cell surface staining. 3 conditions were evaluated: no barcode/no permeabilization, permeabilization only/no barcoding and permeabilization followed by barcoding. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The data shown are representative of an independent experiment. Experiments were performed 3 times independently. PBMC from different individuals were used for each independent experiment.

    Techniques Used: Expressing, Staining

    Effects of different buffers on the detection of cell surface markers. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). Here, we show the effects of different permeabilization conditions on the frequency of various cell surface markers. a.) Histograms showing the frequency and distribution of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events in the CS condition. The concentrations of antibodies used were: CD45 (0.5 mg/ml), CD19 (0.5 mg/ml), CD3 (0.08 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). b.) Comparison of the frequency of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events between the CS condition and the different permeabilization conditions. Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Effects of different buffers on the detection of cell surface markers. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). Here, we show the effects of different permeabilization conditions on the frequency of various cell surface markers. a.) Histograms showing the frequency and distribution of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events in the CS condition. The concentrations of antibodies used were: CD45 (0.5 mg/ml), CD19 (0.5 mg/ml), CD3 (0.08 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). b.) Comparison of the frequency of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events between the CS condition and the different permeabilization conditions. Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Staining

    8) Product Images from "STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells"

    Article Title: STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201911592

    STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P
    Figure Legend Snippet: STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P

    Techniques Used: Mutagenesis, Flow Cytometry, Staining

    9) Product Images from "HPV16 drives cancer immune escape via NLRX1-mediated degradation of STING"

    Article Title: HPV16 drives cancer immune escape via NLRX1-mediated degradation of STING

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI129497

    HPV16 E7 specifically interacts with NLRX1. ( A and B ) 93VU147T and SCC90 cells were lysed, precleared, and incubated with an isotype control antibody and anti-HPV16 E7. Immunoprecipitation was performed using Protein A/G UltraLink Resin, and immunoprecipitated protein complexes were washed before SDS-PAGE. Immunoblotting of NLRX1 and specificity control proteins was carried out. ( C ) The whole-cell lysates of HPV18 E7–expressing UMSCC49 cells were precleared and incubated with IgG2a isotype control or anti–HPV18 E7, followed by incubation with Protein A/G UltraLink Resin for 2 hours at room temperature. Immunoprecipitated protein complexes were washed and subjected to SDS-PAGE. Immunoblotting of STING and specificity control proteins was performed. Experiments were performed 3 times, and representative results are shown. ( D ) 93VU147T cells were stained with MitoTracker, followed by fixation, permeabilization, and staining with NLRX1 and HPV16 E7. Nuclei were counterstained with Hoechst. Representative images and colocalization overlay are shown (scale bars: 10 μm). Experiments were performed twice.
    Figure Legend Snippet: HPV16 E7 specifically interacts with NLRX1. ( A and B ) 93VU147T and SCC90 cells were lysed, precleared, and incubated with an isotype control antibody and anti-HPV16 E7. Immunoprecipitation was performed using Protein A/G UltraLink Resin, and immunoprecipitated protein complexes were washed before SDS-PAGE. Immunoblotting of NLRX1 and specificity control proteins was carried out. ( C ) The whole-cell lysates of HPV18 E7–expressing UMSCC49 cells were precleared and incubated with IgG2a isotype control or anti–HPV18 E7, followed by incubation with Protein A/G UltraLink Resin for 2 hours at room temperature. Immunoprecipitated protein complexes were washed and subjected to SDS-PAGE. Immunoblotting of STING and specificity control proteins was performed. Experiments were performed 3 times, and representative results are shown. ( D ) 93VU147T cells were stained with MitoTracker, followed by fixation, permeabilization, and staining with NLRX1 and HPV16 E7. Nuclei were counterstained with Hoechst. Representative images and colocalization overlay are shown (scale bars: 10 μm). Experiments were performed twice.

    Techniques Used: Incubation, Immunoprecipitation, SDS Page, Expressing, Staining

    10) Product Images from "Preferred endocytosis of amyloid precursor protein from cholesterol-enriched lipid raft microdomains"

    Article Title: Preferred endocytosis of amyloid precursor protein from cholesterol-enriched lipid raft microdomains

    Journal: bioRxiv

    doi: 10.1101/2020.06.26.172874

    Cellular cholesterol levels determined the localization of endogenous APP in lipid raft microdomains from rat primary hippocampal neurons. A. Rat primary hippocampal neurons (DIV21-DIV23) were incubated with either 1.5 mM MβCD-cholesterol or 2 mM MβCD. Then, cells were incubated with filipin to stain free cholesterol levels. Representative confocal image are shown. Data are representative of three independent experiments. Scare bars correspond to 10 μm. B. Filipin fluorescent intensities (n=3). C. Hippocampal neurons were treated with MβCD-cholesterol or MβCD, followed by incubation with APP antibody and cholera toxin B (CTB) at 4°C. After fixing and permeabilization, neurons were incubated with NeuN antibody to detect neurons. Confocal image from four independent experiments is shown. Scare bars correspond to 10 μm. D. Co-efficiency of APP and CTB was analyzed with Image J (n=4). Statistical analysis was performed by one-way ANOVA: ***p
    Figure Legend Snippet: Cellular cholesterol levels determined the localization of endogenous APP in lipid raft microdomains from rat primary hippocampal neurons. A. Rat primary hippocampal neurons (DIV21-DIV23) were incubated with either 1.5 mM MβCD-cholesterol or 2 mM MβCD. Then, cells were incubated with filipin to stain free cholesterol levels. Representative confocal image are shown. Data are representative of three independent experiments. Scare bars correspond to 10 μm. B. Filipin fluorescent intensities (n=3). C. Hippocampal neurons were treated with MβCD-cholesterol or MβCD, followed by incubation with APP antibody and cholera toxin B (CTB) at 4°C. After fixing and permeabilization, neurons were incubated with NeuN antibody to detect neurons. Confocal image from four independent experiments is shown. Scare bars correspond to 10 μm. D. Co-efficiency of APP and CTB was analyzed with Image J (n=4). Statistical analysis was performed by one-way ANOVA: ***p

    Techniques Used: Incubation, Staining, CtB Assay

    Cellular cholesterol levels altered the accumulation of APP in early endosomes. CHO PS1 WT and PS1 ΔE9 cells were incubated with APP antibody at 4°C and transferred to 37°C for indicated times to allow internalization of the labeled surface APP. Then, cells were fixed and surface APP was stained with anti-mouse IgG secondary antibody to eliminate remaining surface APP signal. Following permeabilization, cells were labeled with EEA1 antibody to label early endosomes. Subsequently, anti-mouse Alexa647 (red)- or anti-rabbit Aexa488 (green)-conjugated secondary antibodies were used to label internalized APP and early endosomes, respectively. A. Representative confocal image showing APP localization at each time. Data are representative of four independent experiments. Scare bars correspond to 10 μm. B. Fluorescence intensities of APP and EEA1 were measured using Image J. The co-efficiency of APP and early endosomes was determined with Image J (n=4). Statistical analysis was analyzed by one-way ANOVA: *** indicates P
    Figure Legend Snippet: Cellular cholesterol levels altered the accumulation of APP in early endosomes. CHO PS1 WT and PS1 ΔE9 cells were incubated with APP antibody at 4°C and transferred to 37°C for indicated times to allow internalization of the labeled surface APP. Then, cells were fixed and surface APP was stained with anti-mouse IgG secondary antibody to eliminate remaining surface APP signal. Following permeabilization, cells were labeled with EEA1 antibody to label early endosomes. Subsequently, anti-mouse Alexa647 (red)- or anti-rabbit Aexa488 (green)-conjugated secondary antibodies were used to label internalized APP and early endosomes, respectively. A. Representative confocal image showing APP localization at each time. Data are representative of four independent experiments. Scare bars correspond to 10 μm. B. Fluorescence intensities of APP and EEA1 were measured using Image J. The co-efficiency of APP and early endosomes was determined with Image J (n=4). Statistical analysis was analyzed by one-way ANOVA: *** indicates P

    Techniques Used: Incubation, Labeling, Staining, Fluorescence

    11) Product Images from "Clathrin inhibitor Pitstop-2 disrupts the nuclear pore complex permeability barrier"

    Article Title: Clathrin inhibitor Pitstop-2 disrupts the nuclear pore complex permeability barrier

    Journal: Scientific Reports

    doi: 10.1038/srep09994

    Pitstop-2 induces reversible collapse of the NPC permeability barrier Representative confocal images of digitonin-permeabilized GM7373 cells at 30 minutes after treatment with 0.1% DMSO ( A ), 30 μM Pitstop-2 ( B ) and 5% CHD ( D ). Washout of Pitstop-2 one hour prior to permeabilization leads to restoration of permeability barrier ( C ). Scale bar = 10 μm. Kinetics of 70 kDa dextran influx into the nuclei of digitonin-permeabilized cells upon treatment with respective compounds ( E ). 20 cells from three separate experiments were analyzed for each condition. Error bars represent standard error of the mean.
    Figure Legend Snippet: Pitstop-2 induces reversible collapse of the NPC permeability barrier Representative confocal images of digitonin-permeabilized GM7373 cells at 30 minutes after treatment with 0.1% DMSO ( A ), 30 μM Pitstop-2 ( B ) and 5% CHD ( D ). Washout of Pitstop-2 one hour prior to permeabilization leads to restoration of permeability barrier ( C ). Scale bar = 10 μm. Kinetics of 70 kDa dextran influx into the nuclei of digitonin-permeabilized cells upon treatment with respective compounds ( E ). 20 cells from three separate experiments were analyzed for each condition. Error bars represent standard error of the mean.

    Techniques Used: Permeability

    12) Product Images from "Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi"

    Article Title: Antimicrobial peptides from Capsicum chinense fruits: agronomic alternatives against phytopathogenic fungi

    Journal: Bioscience Reports

    doi: 10.1042/BSR20200950

    Membrane permeabilization assay Assay performed with fluorescence microscopy of cells of different filamentous fungi treated with the fluorescent probe Sytox green to evaluate membrane permeabilization. Control cells (grown in the absence of fractions) and treated cells with F4 and F5 fractions for 24 h. The assay were performed at the concentration of 200 µg.ml −1 and the cells were visualized by DIC and fluorescence; bars = 50 μm.
    Figure Legend Snippet: Membrane permeabilization assay Assay performed with fluorescence microscopy of cells of different filamentous fungi treated with the fluorescent probe Sytox green to evaluate membrane permeabilization. Control cells (grown in the absence of fractions) and treated cells with F4 and F5 fractions for 24 h. The assay were performed at the concentration of 200 µg.ml −1 and the cells were visualized by DIC and fluorescence; bars = 50 μm.

    Techniques Used: Fluorescence, Microscopy, Concentration Assay

    13) Product Images from "Localization to the Cortical Cytoskeleton Is Necessary for Nf2/Merlin-Dependent Epidermal Growth Factor Receptor Silencing ▿Localization to the Cortical Cytoskeleton Is Necessary for Nf2/Merlin-Dependent Epidermal Growth Factor Receptor Silencing ▿ †"

    Article Title: Localization to the Cortical Cytoskeleton Is Necessary for Nf2/Merlin-Dependent Epidermal Growth Factor Receptor Silencing ▿Localization to the Cortical Cytoskeleton Is Necessary for Nf2/Merlin-Dependent Epidermal Growth Factor Receptor Silencing ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01139-07

    The N terminus directs merlin to an insoluble apical network. Simultaneous fixation and permeabilization (fix 2) revealed that Nf2 wt (A) and ezrin (B) decorate the same cortical network in LDCs. Merge of the images is shown in panel C; lower magnification of images in panels A and B are represented in panels E and F, respectively. (D) Nf2 18-595 exhibits a fragmented localization to this network (see also Fig. S2D in the supplemental material). (E) The network localization of ezrin is not altered in the presence or absence of Nf2 wt (E and F). (G) LDCs expressing Nf2 wt were treated with jasplakinolide (2 μM, 1 h, 37°C). Jasplakinolide treatment yielded markedly enlarged compartments within the Nf2 wt -decorated cortical actin network. Upon detergent extraction prior to fixation (fix 3), Nf2 wt (H), but not ezrin (I) or Nf2 18-595 (J), retained network staining. (K) Immunofluorescence localization of NHE-RF1 in LDCs fixed and then permeabilized (fix 1) revealed that NHE-RF1 localizes to a similar cortical network. (L) Confocal imaging of confluent LDCs expressing various versions of Nf2 (fix 1) revealed that Nf2 wt colocalizes with apically distributed concanavalin A (ConA; a lectin that binds polysaccharides on the cell surface) and ezrin (an established apical marker); Nf2 wt is also enriched at cell-cell boundaries (arrowheads). However, careful examination revealed that Nf2 18-595 is not retained at the apical compartment and instead extends basally; Nf2 18-595 also fails to be enriched at cell-cell junctions (arrowheads). Little overlap is seen between apically distributed Nf2 wt and the basal marker, paxillin; in contrast, Nf2 18-595 does overlap with paxillin. Nuclei in panel L are stained with TOTO 3 (blue). Bars = 5 μm. n ≥ 3.
    Figure Legend Snippet: The N terminus directs merlin to an insoluble apical network. Simultaneous fixation and permeabilization (fix 2) revealed that Nf2 wt (A) and ezrin (B) decorate the same cortical network in LDCs. Merge of the images is shown in panel C; lower magnification of images in panels A and B are represented in panels E and F, respectively. (D) Nf2 18-595 exhibits a fragmented localization to this network (see also Fig. S2D in the supplemental material). (E) The network localization of ezrin is not altered in the presence or absence of Nf2 wt (E and F). (G) LDCs expressing Nf2 wt were treated with jasplakinolide (2 μM, 1 h, 37°C). Jasplakinolide treatment yielded markedly enlarged compartments within the Nf2 wt -decorated cortical actin network. Upon detergent extraction prior to fixation (fix 3), Nf2 wt (H), but not ezrin (I) or Nf2 18-595 (J), retained network staining. (K) Immunofluorescence localization of NHE-RF1 in LDCs fixed and then permeabilized (fix 1) revealed that NHE-RF1 localizes to a similar cortical network. (L) Confocal imaging of confluent LDCs expressing various versions of Nf2 (fix 1) revealed that Nf2 wt colocalizes with apically distributed concanavalin A (ConA; a lectin that binds polysaccharides on the cell surface) and ezrin (an established apical marker); Nf2 wt is also enriched at cell-cell boundaries (arrowheads). However, careful examination revealed that Nf2 18-595 is not retained at the apical compartment and instead extends basally; Nf2 18-595 also fails to be enriched at cell-cell junctions (arrowheads). Little overlap is seen between apically distributed Nf2 wt and the basal marker, paxillin; in contrast, Nf2 18-595 does overlap with paxillin. Nuclei in panel L are stained with TOTO 3 (blue). Bars = 5 μm. n ≥ 3.

    Techniques Used: Expressing, Staining, Immunofluorescence, Imaging, Marker

    14) Product Images from "Mechanisms of Action of Substituted β-Amino Alkanols on Leishmania donovani"

    Article Title: Mechanisms of Action of Substituted β-Amino Alkanols on Leishmania donovani

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.04003-14

    Assessment of plasma membrane permeabilization in L. donovani promastigotes by β-amino alkanols. Promastigotes were incubated under standard conditions with selected amino alkanols at equipotent concentrations corresponding to their IC 90 s in HBSS-Glc.
    Figure Legend Snippet: Assessment of plasma membrane permeabilization in L. donovani promastigotes by β-amino alkanols. Promastigotes were incubated under standard conditions with selected amino alkanols at equipotent concentrations corresponding to their IC 90 s in HBSS-Glc.

    Techniques Used: Incubation, Gas Chromatography

    15) Product Images from "Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves"

    Article Title: Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20082129

    The anatomical position of aortic DCs in aortic wall. After immunostaining the aortic sheet using CD31 antibody to visualize the endothelium, confocal images were taken along the Z axis to visualize all dendritic processes from each EYFP + DC and were reconstituted to a Z stack. (A) Reconstituted Z planes from aortic valve (top), aortic sinus (middle), and aortic arch (bottom) show that the aortic DCs are localized beneath the endothelium. A small number of DCs in the aortic sinus extend their processes into the vessel lumen (A, middle, arrow). (B) Three-dimensional images were reconstituted using the Imaris program to better visualize the position of aortic DCs. Arrows indicate the vessel lumen side. (C) Exposure of some dendritic processes into the lumen. The aortic sheets from EYFP transgenic mice were stained with CD11c antibody without tissue permeabilization to see whether dendritic processes could capture antibody from the lumen. The confocal images were reconstituted in three dimensions. (D) Biotinylated anti-CD11c and hamster IgG control antibody were injected i.v. to CD11c-EYFP mice. After 3 h, the aortic segments were isolated, incubated with horseradish peroxidase–conjugated streptavidin, and the stained CD11c was enhanced with Alexa Fluor 555 tyramide. (E) The elastic lamina was visualized using its autofluorescence. The confocal images were reconstituted in three dimensions. Each figure is representative of at least three experiments. Bars, 20 µm.
    Figure Legend Snippet: The anatomical position of aortic DCs in aortic wall. After immunostaining the aortic sheet using CD31 antibody to visualize the endothelium, confocal images were taken along the Z axis to visualize all dendritic processes from each EYFP + DC and were reconstituted to a Z stack. (A) Reconstituted Z planes from aortic valve (top), aortic sinus (middle), and aortic arch (bottom) show that the aortic DCs are localized beneath the endothelium. A small number of DCs in the aortic sinus extend their processes into the vessel lumen (A, middle, arrow). (B) Three-dimensional images were reconstituted using the Imaris program to better visualize the position of aortic DCs. Arrows indicate the vessel lumen side. (C) Exposure of some dendritic processes into the lumen. The aortic sheets from EYFP transgenic mice were stained with CD11c antibody without tissue permeabilization to see whether dendritic processes could capture antibody from the lumen. The confocal images were reconstituted in three dimensions. (D) Biotinylated anti-CD11c and hamster IgG control antibody were injected i.v. to CD11c-EYFP mice. After 3 h, the aortic segments were isolated, incubated with horseradish peroxidase–conjugated streptavidin, and the stained CD11c was enhanced with Alexa Fluor 555 tyramide. (E) The elastic lamina was visualized using its autofluorescence. The confocal images were reconstituted in three dimensions. Each figure is representative of at least three experiments. Bars, 20 µm.

    Techniques Used: Immunostaining, Transgenic Assay, Mouse Assay, Staining, Injection, Isolation, Incubation

    16) Product Images from "Selective Phosphorylation of the Dlg1AB Variant Is Critical for TCR-Induced p38 Activation and Induction of Proinflammatory Cytokines in CD8+ T Cells"

    Article Title: Selective Phosphorylation of the Dlg1AB Variant Is Critical for TCR-Induced p38 Activation and Induction of Proinflammatory Cytokines in CD8+ T Cells

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1401196

    Acute knockout of Dlg1 impairs p38 activation and NFAT-dependent proinflammatory cytokines gene expression and target cell lysis in response to TCR stimulation. ( A ) Genomic organization of Dlg1 flox/flox ). F and R refer to the location of the forward and reverse primers for gDNA analysis. ( B – L ) CD8 + T cells were isolated from spleens of OT-1 Dlg1 flox/flox mice and expanded on plate-bound anti-CD3 and anti-CD28 Ab for 48–72 h, followed by infection with retroviral cre recombinase or vector control. (B) Genomic DNA was isolated, and PCR analysis was performed. (C) Whole-cell lysates were subjected to SDS-PAGE and blotted with anti-Dlg1 or anti-p38. (D) Cells were stimulated with plate-bound anti-CD3 and anti-CD28 Ab for 15 min, followed by fixation, permeabilization, and staining for p-p38 180/182. Error bars represent SD of three independent experiments. (E–J) Cells were left unstimulated or stimulated with plate-bound anti-CD3 and anti-CD28 for 2 h (E and F) or 6 h (G–J), followed by mRNA isolation, reverse transcription, and qPCR analysis using primers specific for NFATc1(E), IκBα (F), IFN-γ (G), TNF-α (H), IL-2 (I), or granzyme B (J). All values were normalized to L32 and unstimulated values set to 1.0. Error bars represent stand deviation of triplicates; data are representative of four independent experiments. (K and L) Cells were incubated with EG.7 thymoma cells at indicated ratios for 2 h, following surface staining for CD107a (K) or lactate dehydrogenase cytotoxicity assay (L). ** p
    Figure Legend Snippet: Acute knockout of Dlg1 impairs p38 activation and NFAT-dependent proinflammatory cytokines gene expression and target cell lysis in response to TCR stimulation. ( A ) Genomic organization of Dlg1 flox/flox ). F and R refer to the location of the forward and reverse primers for gDNA analysis. ( B – L ) CD8 + T cells were isolated from spleens of OT-1 Dlg1 flox/flox mice and expanded on plate-bound anti-CD3 and anti-CD28 Ab for 48–72 h, followed by infection with retroviral cre recombinase or vector control. (B) Genomic DNA was isolated, and PCR analysis was performed. (C) Whole-cell lysates were subjected to SDS-PAGE and blotted with anti-Dlg1 or anti-p38. (D) Cells were stimulated with plate-bound anti-CD3 and anti-CD28 Ab for 15 min, followed by fixation, permeabilization, and staining for p-p38 180/182. Error bars represent SD of three independent experiments. (E–J) Cells were left unstimulated or stimulated with plate-bound anti-CD3 and anti-CD28 for 2 h (E and F) or 6 h (G–J), followed by mRNA isolation, reverse transcription, and qPCR analysis using primers specific for NFATc1(E), IκBα (F), IFN-γ (G), TNF-α (H), IL-2 (I), or granzyme B (J). All values were normalized to L32 and unstimulated values set to 1.0. Error bars represent stand deviation of triplicates; data are representative of four independent experiments. (K and L) Cells were incubated with EG.7 thymoma cells at indicated ratios for 2 h, following surface staining for CD107a (K) or lactate dehydrogenase cytotoxicity assay (L). ** p

    Techniques Used: Knock-Out, Activation Assay, Expressing, Lysis, Isolation, Mouse Assay, Infection, Plasmid Preparation, Polymerase Chain Reaction, SDS Page, Staining, Real-time Polymerase Chain Reaction, Incubation, Cytotoxicity Assay

    17) Product Images from "Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin"

    Article Title: Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

    Journal: Toxins

    doi: 10.3390/toxins12050343

    Chitosan controls the lysenin-mediated transport of PI across the membrane of Jurkat cells. PI did not cross the cell membrane in the absence of lysenin, as indicated by the steady fluorescence intensity. In contrast, a continual increase in fluorescence, indicative of PI intercalation, was observed after lysenin addition (10 nM final concentration). For a similarly permeabilized sample, addition of chitosan 20 min. after the initiation of permeabilization quickly stabilized the fluorescent intensity and indicated that PI was prevented from further crossing the cell membrane. The symbols represent average values from four experiments (n = 4, ±SD) that comprised individual samples made from the same cell culture batch.
    Figure Legend Snippet: Chitosan controls the lysenin-mediated transport of PI across the membrane of Jurkat cells. PI did not cross the cell membrane in the absence of lysenin, as indicated by the steady fluorescence intensity. In contrast, a continual increase in fluorescence, indicative of PI intercalation, was observed after lysenin addition (10 nM final concentration). For a similarly permeabilized sample, addition of chitosan 20 min. after the initiation of permeabilization quickly stabilized the fluorescent intensity and indicated that PI was prevented from further crossing the cell membrane. The symbols represent average values from four experiments (n = 4, ±SD) that comprised individual samples made from the same cell culture batch.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    18) Product Images from "An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation"

    Article Title: An EdU-based flow cytometry assay to evaluate chicken T lymphocyte proliferation

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-020-02433-0

    Effect of permeabilization reagents on the detection of EdU + cells. Mononuclear splenocyte cells, cultured for 72 h in the presence or absence of 1 μg/ml ConA, were fixed and treated with different permeabilization reagents (saponin or Triton X-100). ( a ) Flow cytometry analysis for detecting EdU incorporation into cells. ( b ) Representing dot plots of the cells treated with different permeabilization reagents. ( c ) Comparison of median fluorescence intensity (MFI) in EdU − cells (autofluorescence) treated with saponin or Triton X-100. ( d ) Comparison of MFI of EdU + cells treated with 0.5% or 0.2% saponin. Results are the mean ± standard deviation from of 2 independent experiments, performed in duplicate. In each experiment, the cells of one chicken were analyzed. Per sample, 20,000 events were acquired on a FACSMelody flow cytometer
    Figure Legend Snippet: Effect of permeabilization reagents on the detection of EdU + cells. Mononuclear splenocyte cells, cultured for 72 h in the presence or absence of 1 μg/ml ConA, were fixed and treated with different permeabilization reagents (saponin or Triton X-100). ( a ) Flow cytometry analysis for detecting EdU incorporation into cells. ( b ) Representing dot plots of the cells treated with different permeabilization reagents. ( c ) Comparison of median fluorescence intensity (MFI) in EdU − cells (autofluorescence) treated with saponin or Triton X-100. ( d ) Comparison of MFI of EdU + cells treated with 0.5% or 0.2% saponin. Results are the mean ± standard deviation from of 2 independent experiments, performed in duplicate. In each experiment, the cells of one chicken were analyzed. Per sample, 20,000 events were acquired on a FACSMelody flow cytometer

    Techniques Used: Cell Culture, Flow Cytometry, Fluorescence, Standard Deviation

    19) Product Images from "Performance Evaluation of Fast Microfluidic Thermal Lysis of Bacteria for Diagnostic Sample Preparation †"

    Article Title: Performance Evaluation of Fast Microfluidic Thermal Lysis of Bacteria for Diagnostic Sample Preparation †

    Journal: Diagnostics

    doi: 10.3390/diagnostics3010105

    Silicon and glass microfluidic lysis chip—layout in Tanner L-edit software ( top ) and finished device ( bottom ). The top device has a 1 mm wide channel and the bottom device an 0.5 mm wide channel. The external chip dimensions are 70.5 mm × 9 mm × 1 mm. The rectangle marked “ROI” denotes the imaging area used for the BacLight live:dead membrane permeabilization assay (see Section 2.6 ).
    Figure Legend Snippet: Silicon and glass microfluidic lysis chip—layout in Tanner L-edit software ( top ) and finished device ( bottom ). The top device has a 1 mm wide channel and the bottom device an 0.5 mm wide channel. The external chip dimensions are 70.5 mm × 9 mm × 1 mm. The rectangle marked “ROI” denotes the imaging area used for the BacLight live:dead membrane permeabilization assay (see Section 2.6 ).

    Techniques Used: Lysis, Chromatin Immunoprecipitation, Software, Imaging

    20) Product Images from "The Hyphal-Associated Adhesin and Invasin Als3 of Candida albicans Mediates Iron Acquisition from Host Ferritin"

    Article Title: The Hyphal-Associated Adhesin and Invasin Als3 of Candida albicans Mediates Iron Acquisition from Host Ferritin

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000217

    C. albicans hyphae invading oral epithelial cells bind ferritin. C. albicans wild-type (SC5314), Δals3 mutant and Δals3+ALS3 re-integrant cells were co-incubated with ferritin-enriched oral epithelial cells and differentially stained. (A), (E), (I) and (M); staining of extracellular (non-invaded) C. albicans with concanavalin A conjugated with fluorescein before cell permeabilization. (B), (F), (J) and (N); calcofluor white staining of whole C. albicans cells following epithelial cell permeabilization. (C), (G), (K) and (O); fluorescent dye (DY649) coupled antibody staining of ferritin. White arrows indicate hyphae surrounded by epithelial ferritin. (D), (H), (L) and (P); merged images. Bar in (P) indicates 10 µm.
    Figure Legend Snippet: C. albicans hyphae invading oral epithelial cells bind ferritin. C. albicans wild-type (SC5314), Δals3 mutant and Δals3+ALS3 re-integrant cells were co-incubated with ferritin-enriched oral epithelial cells and differentially stained. (A), (E), (I) and (M); staining of extracellular (non-invaded) C. albicans with concanavalin A conjugated with fluorescein before cell permeabilization. (B), (F), (J) and (N); calcofluor white staining of whole C. albicans cells following epithelial cell permeabilization. (C), (G), (K) and (O); fluorescent dye (DY649) coupled antibody staining of ferritin. White arrows indicate hyphae surrounded by epithelial ferritin. (D), (H), (L) and (P); merged images. Bar in (P) indicates 10 µm.

    Techniques Used: Mutagenesis, Incubation, Staining

    21) Product Images from "Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag"

    Article Title: Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag

    Journal: Virology Journal

    doi: 10.1186/1743-422X-5-23

    SDF-1 induced CXCR4 downregulation is TSG101-dependent . (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.
    Figure Legend Snippet: SDF-1 induced CXCR4 downregulation is TSG101-dependent . (A) COS-1 cells co-transfected with HA-tagged CXCR4 and either GFP, TSG101-GFP, or siRNA directed against TSG101, as indicated, were incubated with an anti-HA antibody for 1 hour on ice. Cells were then either fixed and stained with a secondary antibody to detect cell surface CXCR4 (first row), or incubated in DMEM/10% FBS without SDF (second row) or with 100 nM SDF (third and fourth rows) for 3 hours at 37°C prior to fixation, permeabilization and secondary-antibody staining. The cells were then analysed for GFP fluorescence (green), or CXCR4 expression (red). Blue represents nuclear staining. Scale bars = 10 μm. (B) Immunoblot of COS-1 cells co-transfected with HA-tagged CXCR4 and siRNA directed against either TSG101 or LacZ. Top panel, TSG101 Western blot; bottom panel, actin Western blot.

    Techniques Used: Transfection, Incubation, Staining, Fluorescence, Expressing, Western Blot

    22) Product Images from "The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells"

    Article Title: The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4+ T cells

    Journal: Nature Communications

    doi: 10.1038/ncomms1272

    Nr4a2 stabilizes Foxp3 expression in Tregs. ( a ) Top: CD4 + and CD8 + compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. ( b ) Quantification of the results in a . Ratio of YFP + /YFP – in total Foxp3 + cells. Filled bars: results of thymus; open bars: results of spleen plus lymph nodes. Data are pooled from three independent experiments, with six mice from each genotype total, aged 11- to 13 weeks (mean±s.d.). ( c ) Left: CFSE-labeled CD4 + CD25 + YFP + Tregs from Nr4a2 +/+ Foxp3 Yfp-Cre/+ or Nr4a2 fl/fl Foxp3 Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml −1 IL-2, thymidine (2 mM) and z-VAD-fmk (10 μM) were analysed for Foxp3 expression at the time indicated. YFP-fluorescences were bleached by fixation/permeabilization. Right: quantification of the results in left. Open squares: Nr4a2 fl/fl Foxp3 Yfp-Cre cells; crosses: Nr4a2 +/+ Foxp3 Yfp-Cre cells. ( d ) Proliferation and survival status of cells in c . Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2 fl/fl Foxp3 Yfp-Cre cells; crosses: Nr4a2 +/+ Foxp3 Yfp-Cre cells. ( e ) Rag2 – mice were co-transferred with 3×10 5 CD4 + CD25 + YFP + Ly5.2 + Tregs from Nr4a2 fl/fl Foxp3 Yfp-Cre mice and 3×10 5 CD4 + CD25 + Ly5.1 + wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. ( f ) Quantification of the results in e . Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3 + fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1 + fractions; open bars: cells in Ly5.1 − fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). ( g ) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4 + T cells, or of naïve CD4 + T cells stimulated under the iTreg condition. ( h ) Left: coimmunoprecipitation of Flag-Runx1 and T7-Nr4a in 293T cell lysates. Right: quantification of the result in left. Intensity of the anti-T7 blot of the anti-Flag immunoprecipitates, normalized with the anti-T7 input. ( i ) Top: schematic representation of the reporter construct. Nr4a-binding site and the Runx1-binding sites are indicated. Bottom: results of the luciferase assay. Numbers in FACS plots represent percentages of cells in the gated area. Data are representative of three ( c , h , i ) or two ( d ) independent experiments (mean±s.d. of triplicate). * P
    Figure Legend Snippet: Nr4a2 stabilizes Foxp3 expression in Tregs. ( a ) Top: CD4 + and CD8 + compartments of whole cells from the indicated organs. Bottom: Foxp3 and YFP expression in CD4-SP fractions in top. ( b ) Quantification of the results in a . Ratio of YFP + /YFP – in total Foxp3 + cells. Filled bars: results of thymus; open bars: results of spleen plus lymph nodes. Data are pooled from three independent experiments, with six mice from each genotype total, aged 11- to 13 weeks (mean±s.d.). ( c ) Left: CFSE-labeled CD4 + CD25 + YFP + Tregs from Nr4a2 +/+ Foxp3 Yfp-Cre/+ or Nr4a2 fl/fl Foxp3 Yfp-Cre/+ mice, cultured under neutral condition with 20 ng ml −1 IL-2, thymidine (2 mM) and z-VAD-fmk (10 μM) were analysed for Foxp3 expression at the time indicated. YFP-fluorescences were bleached by fixation/permeabilization. Right: quantification of the results in left. Open squares: Nr4a2 fl/fl Foxp3 Yfp-Cre cells; crosses: Nr4a2 +/+ Foxp3 Yfp-Cre cells. ( d ) Proliferation and survival status of cells in c . Left: CFSE dilution of live cells at 144 h. Right: live cell number at indicated time points. Open squares: Nr4a2 fl/fl Foxp3 Yfp-Cre cells; crosses: Nr4a2 +/+ Foxp3 Yfp-Cre cells. ( e ) Rag2 – mice were co-transferred with 3×10 5 CD4 + CD25 + YFP + Ly5.2 + Tregs from Nr4a2 fl/fl Foxp3 Yfp-Cre mice and 3×10 5 CD4 + CD25 + Ly5.1 + wild-type Tregs. Foxp3 expression at day 0 and days 20 after transfer are shown. ( f ) Quantification of the results in e . Left: mean fluorescence intensity (MFI) of Foxp3-staining of the Foxp3 + fractions. Right: ratio of Foxp3-positive fractions. Closed bars: cells in Ly5.1 + fractions; open bars: cells in Ly5.1 − fractions. Data are pooled from two independent experiments with five mice from each sample set total (mean±s.d.). ( g ) Coimmunoprecipitation of Nr4a2 and Runx1 from total cell lysates of naïve CD4 + T cells, or of naïve CD4 + T cells stimulated under the iTreg condition. ( h ) Left: coimmunoprecipitation of Flag-Runx1 and T7-Nr4a in 293T cell lysates. Right: quantification of the result in left. Intensity of the anti-T7 blot of the anti-Flag immunoprecipitates, normalized with the anti-T7 input. ( i ) Top: schematic representation of the reporter construct. Nr4a-binding site and the Runx1-binding sites are indicated. Bottom: results of the luciferase assay. Numbers in FACS plots represent percentages of cells in the gated area. Data are representative of three ( c , h , i ) or two ( d ) independent experiments (mean±s.d. of triplicate). * P

    Techniques Used: Expressing, Mouse Assay, Labeling, Cell Culture, Fluorescence, Staining, Construct, Binding Assay, Luciferase, FACS

    23) Product Images from "MicroRNA 199a-5p Attenuates Retrograde Transport and Protects against Toxin-Induced Inhibition of Protein Biosynthesis"

    Article Title: MicroRNA 199a-5p Attenuates Retrograde Transport and Protects against Toxin-Induced Inhibition of Protein Biosynthesis

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00548-17

    miR-199a-5p regulates M6PR plasma membrane expression and M6PR intracellular transport. (A) Immunofluorescence analysis showing steady-state localization of endogenous M6PR and Vps26A after 48 h of transfection with CM and miR-199a-5p. Scale bar = 25 μm. Magnif, magnification of areas in dotted boxes. Scale bar = 5 μm. (B) Representative flow cytometry histograms of total M6PR staining (top) and extracellular M6PR (bottom). Right, quantification of geometric mean fluorescence. AU, arbitrary units. (C) Immunofluorescence analysis showing HeLa cells transfected with CM or miR-199a-5p as indicated and incubated with anti-M6PR antibody for 60 min at 4°C and then either fixed and stained with anti-mouse Alexa Fluor 488-conjugated M6PR antibody without Triton X-100 (Tx-100; top panels) or allowed to internalize antibody complexes for 60 min at 37°C, fixed with PFA, and stained with anti-mouse Alexa Fluor 488-conjugated antibody (bottom panels). The Tx-100 permeabilization step was included to visualize internal compartments. To visualize the F-actin fibers and nuclei, phalloidin red and DAPI were used, respectively. Scale bar = 25 μm.
    Figure Legend Snippet: miR-199a-5p regulates M6PR plasma membrane expression and M6PR intracellular transport. (A) Immunofluorescence analysis showing steady-state localization of endogenous M6PR and Vps26A after 48 h of transfection with CM and miR-199a-5p. Scale bar = 25 μm. Magnif, magnification of areas in dotted boxes. Scale bar = 5 μm. (B) Representative flow cytometry histograms of total M6PR staining (top) and extracellular M6PR (bottom). Right, quantification of geometric mean fluorescence. AU, arbitrary units. (C) Immunofluorescence analysis showing HeLa cells transfected with CM or miR-199a-5p as indicated and incubated with anti-M6PR antibody for 60 min at 4°C and then either fixed and stained with anti-mouse Alexa Fluor 488-conjugated M6PR antibody without Triton X-100 (Tx-100; top panels) or allowed to internalize antibody complexes for 60 min at 37°C, fixed with PFA, and stained with anti-mouse Alexa Fluor 488-conjugated antibody (bottom panels). The Tx-100 permeabilization step was included to visualize internal compartments. To visualize the F-actin fibers and nuclei, phalloidin red and DAPI were used, respectively. Scale bar = 25 μm.

    Techniques Used: Expressing, Immunofluorescence, Transfection, Flow Cytometry, Cytometry, Staining, Fluorescence, Incubation

    24) Product Images from "Non-Thermal Plasma Application in Tumor-Bearing Mice Induces Increase of Serum HMGB1"

    Article Title: Non-Thermal Plasma Application in Tumor-Bearing Mice Induces Increase of Serum HMGB1

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21145128

    CAP-dependent translocation of CRT and HSP70. ( a ) The scheme of the analysis of CRT and HSP−70 translocation to the outer cellular membrane. MX−7 cells were irradiated by CAP for 1 min and 24 h after ecto-CRT and ecto-HSP70 were analyzed. ( b ) Quantification data for the ecto-CRT and ecto-HSP70 positive cells. ( c – f ) Analysis of the extracellular ( c , d ) and total ( e , f ) CRT and HSP70. MX−7 cells were stained with anti-CRT ( c , e ) and anti-HMGB1 ( d , f ). Staining was performed with ( e , f ) or no ( c , d ) fixation and permeabilization. Representative graphs of three independent repeats.
    Figure Legend Snippet: CAP-dependent translocation of CRT and HSP70. ( a ) The scheme of the analysis of CRT and HSP−70 translocation to the outer cellular membrane. MX−7 cells were irradiated by CAP for 1 min and 24 h after ecto-CRT and ecto-HSP70 were analyzed. ( b ) Quantification data for the ecto-CRT and ecto-HSP70 positive cells. ( c – f ) Analysis of the extracellular ( c , d ) and total ( e , f ) CRT and HSP70. MX−7 cells were stained with anti-CRT ( c , e ) and anti-HMGB1 ( d , f ). Staining was performed with ( e , f ) or no ( c , d ) fixation and permeabilization. Representative graphs of three independent repeats.

    Techniques Used: Translocation Assay, Irradiation, Staining

    25) Product Images from "Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin"

    Article Title: Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin

    Journal: Toxins

    doi: 10.3390/toxins12050343

    Chitosan controls the lysenin-mediated transport of PI across the membrane of Jurkat cells. PI did not cross the cell membrane in the absence of lysenin, as indicated by the steady fluorescence intensity. In contrast, a continual increase in fluorescence, indicative of PI intercalation, was observed after lysenin addition (10 nM final concentration). For a similarly permeabilized sample, addition of chitosan 20 min. after the initiation of permeabilization quickly stabilized the fluorescent intensity and indicated that PI was prevented from further crossing the cell membrane. The symbols represent average values from four experiments (n = 4, ±SD) that comprised individual samples made from the same cell culture batch.
    Figure Legend Snippet: Chitosan controls the lysenin-mediated transport of PI across the membrane of Jurkat cells. PI did not cross the cell membrane in the absence of lysenin, as indicated by the steady fluorescence intensity. In contrast, a continual increase in fluorescence, indicative of PI intercalation, was observed after lysenin addition (10 nM final concentration). For a similarly permeabilized sample, addition of chitosan 20 min. after the initiation of permeabilization quickly stabilized the fluorescent intensity and indicated that PI was prevented from further crossing the cell membrane. The symbols represent average values from four experiments (n = 4, ±SD) that comprised individual samples made from the same cell culture batch.

    Techniques Used: Fluorescence, Concentration Assay, Cell Culture

    26) Product Images from "Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells"

    Article Title: Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.286914

    Additional technical and computational validation of ASAP-seq workflows. a. PBMCs were stained with fluorophore-conjugated antibodies and subjected to the ASAP-seq workflow with samples withdrawn at the indicated steps and assessed for fluorophore intensity by flow cytometry. CD3 (top) and CD19 (bottom) signal on fixed cells is hardly affected by permeabilization alone, but after the 37℃ incubation for 1h to mimic the Tn5 transposition reaction, some signal reduction is observed. b. Barcoding scheme of TSA tags using the bridge oligo for TotalSeq TM -A (BOA). TSA tags do not contain UMIs, so to allow molecule counting, UBIs (N9V) are incorporated via the bridge oligo. c. Species mixing experiment as in Figure 1c , using the Post-SPRI approach for tag recovery. Points are colored based on species classification using ATAC fragments. d. ATAC library complexity and TSS enrichment for fragments from each species under the two protein-tag library approaches. e. Comparison of protein tag complexity between libraries prepared using the pre- and post-SPRI approach. f. Comparison of ATAC library complexity between mtscATAC-seq and ASAP-seq. g. Two-dimensional embedding of the PBMC hashing data using t -SNE. The four major clusters (black) correspond to the four hashing antibodies used to stain the PBMCs. 13,772 cells were recovered and1,396 doublets (red) were detected. h. UMAP embedding resolving PBMC cell types based on chromatin accessibility for cells processed by mtscATAC-seq and ASAP-seq. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. i. Selected protein markers (left) and corresponding gene score activities (right) superimposed on the ATAC-clustered PBMCs (for the ASAP-seq sample) as in ( h ).
    Figure Legend Snippet: Additional technical and computational validation of ASAP-seq workflows. a. PBMCs were stained with fluorophore-conjugated antibodies and subjected to the ASAP-seq workflow with samples withdrawn at the indicated steps and assessed for fluorophore intensity by flow cytometry. CD3 (top) and CD19 (bottom) signal on fixed cells is hardly affected by permeabilization alone, but after the 37℃ incubation for 1h to mimic the Tn5 transposition reaction, some signal reduction is observed. b. Barcoding scheme of TSA tags using the bridge oligo for TotalSeq TM -A (BOA). TSA tags do not contain UMIs, so to allow molecule counting, UBIs (N9V) are incorporated via the bridge oligo. c. Species mixing experiment as in Figure 1c , using the Post-SPRI approach for tag recovery. Points are colored based on species classification using ATAC fragments. d. ATAC library complexity and TSS enrichment for fragments from each species under the two protein-tag library approaches. e. Comparison of protein tag complexity between libraries prepared using the pre- and post-SPRI approach. f. Comparison of ATAC library complexity between mtscATAC-seq and ASAP-seq. g. Two-dimensional embedding of the PBMC hashing data using t -SNE. The four major clusters (black) correspond to the four hashing antibodies used to stain the PBMCs. 13,772 cells were recovered and1,396 doublets (red) were detected. h. UMAP embedding resolving PBMC cell types based on chromatin accessibility for cells processed by mtscATAC-seq and ASAP-seq. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. i. Selected protein markers (left) and corresponding gene score activities (right) superimposed on the ATAC-clustered PBMCs (for the ASAP-seq sample) as in ( h ).

    Techniques Used: Staining, Flow Cytometry, Incubation

    ASAP-seq enables a modular and versatile multi-omics toolkit. a. Schematic of experimental design. PBMCs were stained with TBNK panels of the TSA or TSB format at a 1:1 ratio, followed by fixation and permeabilization under mild (LLL) or strong conditions (OMNI). b. Pairwise comparison of centered log-ratio (CLR) normalized TSA and TSB counts for indicated antibodies under mild lysis conditions (n=4,748 cells). Counts were collapsed for unique molecules using UBIs (TSA panel) or UMIs (TSB panel). c. Distribution of percent of mtDNA fragments retained in the library under the two lysis conditions. d. Comparison of CLR normalized TSA counts for indicated proteins under the two tested lysis conditions. Statistical comparisons are Wilcoxon rank sum test with Bonferroni adjusted p-values (ns = not significant; * p adj
    Figure Legend Snippet: ASAP-seq enables a modular and versatile multi-omics toolkit. a. Schematic of experimental design. PBMCs were stained with TBNK panels of the TSA or TSB format at a 1:1 ratio, followed by fixation and permeabilization under mild (LLL) or strong conditions (OMNI). b. Pairwise comparison of centered log-ratio (CLR) normalized TSA and TSB counts for indicated antibodies under mild lysis conditions (n=4,748 cells). Counts were collapsed for unique molecules using UBIs (TSA panel) or UMIs (TSB panel). c. Distribution of percent of mtDNA fragments retained in the library under the two lysis conditions. d. Comparison of CLR normalized TSA counts for indicated proteins under the two tested lysis conditions. Statistical comparisons are Wilcoxon rank sum test with Bonferroni adjusted p-values (ns = not significant; * p adj

    Techniques Used: Staining, Lysis

    ASAP-seq incorporates protein detection in scATAC-seq workflows. a. Schematic of the cell-processing steps that allow retention and profiling of cell-surface markers jointly with chromatin accessibility. Cells are stained with oligo-conjugated antibodies before fixation, permeabilization and transposition with Tn5. b. In droplets, bridge oligos spiked into the barcoding mix promote templated extension of the antibody tags during the first cycle of amplification rendering them complementary to bead-derived barcoding oligos. Extended antibody tags are subsequently barcoded together with the transposed chromatin fragments. c. Species mixing experiment using the Pre-SPRI approach; number of unique nuclear fragments (left) and protein-tag counts (right) associated with each cell barcode. Points are colored based on species classification using ATAC-derived fragments (97.4% agreement by assignment; all but 1 discrepancy was an errant doublet versus singlet classification) d. TSS enrichment scores of mtscATAC-seq without (left) or with concomitant protein tag capture (right). n indicates the number of cells profiled. e. UMAP showing chromatin accessibility-based clustering of PBMCs stained with a 9-antibody panel, with selected markers highlighted. Color bar: protein tag centered log-ratio (CLR) values. f. Cellular distribution of two most commonly detected mtDNA mutations in the population. Thresholds for + were 5% heteroplasmy based on empirical density.
    Figure Legend Snippet: ASAP-seq incorporates protein detection in scATAC-seq workflows. a. Schematic of the cell-processing steps that allow retention and profiling of cell-surface markers jointly with chromatin accessibility. Cells are stained with oligo-conjugated antibodies before fixation, permeabilization and transposition with Tn5. b. In droplets, bridge oligos spiked into the barcoding mix promote templated extension of the antibody tags during the first cycle of amplification rendering them complementary to bead-derived barcoding oligos. Extended antibody tags are subsequently barcoded together with the transposed chromatin fragments. c. Species mixing experiment using the Pre-SPRI approach; number of unique nuclear fragments (left) and protein-tag counts (right) associated with each cell barcode. Points are colored based on species classification using ATAC-derived fragments (97.4% agreement by assignment; all but 1 discrepancy was an errant doublet versus singlet classification) d. TSS enrichment scores of mtscATAC-seq without (left) or with concomitant protein tag capture (right). n indicates the number of cells profiled. e. UMAP showing chromatin accessibility-based clustering of PBMCs stained with a 9-antibody panel, with selected markers highlighted. Color bar: protein tag centered log-ratio (CLR) values. f. Cellular distribution of two most commonly detected mtDNA mutations in the population. Thresholds for + were 5% heteroplasmy based on empirical density.

    Techniques Used: Staining, Amplification, Derivative Assay

    27) Product Images from "Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells"

    Article Title: Scalable, multimodal profiling of chromatin accessibility and protein levels in single cells

    Journal: bioRxiv

    doi: 10.1101/2020.09.08.286914

    Additional technical and computational validation of ASAP-seq workflows. a. PBMCs were stained with fluorophore-conjugated antibodies and subjected to the ASAP-seq workflow with samples withdrawn at the indicated steps and assessed for fluorophore intensity by flow cytometry. CD3 (top) and CD19 (bottom) signal on fixed cells is hardly affected by permeabilization alone, but after the 37℃ incubation for 1h to mimic the Tn5 transposition reaction, some signal reduction is observed. b. Barcoding scheme of TSA tags using the bridge oligo for TotalSeq TM -A (BOA). TSA tags do not contain UMIs, so to allow molecule counting, UBIs (N9V) are incorporated via the bridge oligo. c. Species mixing experiment as in Figure 1c , using the Post-SPRI approach for tag recovery. Points are colored based on species classification using ATAC fragments. d. ATAC library complexity and TSS enrichment for fragments from each species under the two protein-tag library approaches. e. Comparison of protein tag complexity between libraries prepared using the pre- and post-SPRI approach. f. Comparison of ATAC library complexity between mtscATAC-seq and ASAP-seq. g. Two-dimensional embedding of the PBMC hashing data using t -SNE. The four major clusters (black) correspond to the four hashing antibodies used to stain the PBMCs. 13,772 cells were recovered and1,396 doublets (red) were detected. h. UMAP embedding resolving PBMC cell types based on chromatin accessibility for cells processed by mtscATAC-seq and ASAP-seq. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. i. Selected protein markers (left) and corresponding gene score activities (right) superimposed on the ATAC-clustered PBMCs (for the ASAP-seq sample) as in ( h ).
    Figure Legend Snippet: Additional technical and computational validation of ASAP-seq workflows. a. PBMCs were stained with fluorophore-conjugated antibodies and subjected to the ASAP-seq workflow with samples withdrawn at the indicated steps and assessed for fluorophore intensity by flow cytometry. CD3 (top) and CD19 (bottom) signal on fixed cells is hardly affected by permeabilization alone, but after the 37℃ incubation for 1h to mimic the Tn5 transposition reaction, some signal reduction is observed. b. Barcoding scheme of TSA tags using the bridge oligo for TotalSeq TM -A (BOA). TSA tags do not contain UMIs, so to allow molecule counting, UBIs (N9V) are incorporated via the bridge oligo. c. Species mixing experiment as in Figure 1c , using the Post-SPRI approach for tag recovery. Points are colored based on species classification using ATAC fragments. d. ATAC library complexity and TSS enrichment for fragments from each species under the two protein-tag library approaches. e. Comparison of protein tag complexity between libraries prepared using the pre- and post-SPRI approach. f. Comparison of ATAC library complexity between mtscATAC-seq and ASAP-seq. g. Two-dimensional embedding of the PBMC hashing data using t -SNE. The four major clusters (black) correspond to the four hashing antibodies used to stain the PBMCs. 13,772 cells were recovered and1,396 doublets (red) were detected. h. UMAP embedding resolving PBMC cell types based on chromatin accessibility for cells processed by mtscATAC-seq and ASAP-seq. Data for the two different samples were processed together using cell ranger-atac aggr before dimensionality reduction. i. Selected protein markers (left) and corresponding gene score activities (right) superimposed on the ATAC-clustered PBMCs (for the ASAP-seq sample) as in ( h ).

    Techniques Used: Staining, Flow Cytometry, Incubation

    ASAP-seq enables a modular and versatile multi-omics toolkit. a. Schematic of experimental design. PBMCs were stained with TBNK panels of the TSA or TSB format at a 1:1 ratio, followed by fixation and permeabilization under mild (LLL) or strong conditions (OMNI). b. Pairwise comparison of centered log-ratio (CLR) normalized TSA and TSB counts for indicated antibodies under mild lysis conditions (n=4,748 cells). Counts were collapsed for unique molecules using UBIs (TSA panel) or UMIs (TSB panel). c. Distribution of percent of mtDNA fragments retained in the library under the two lysis conditions. d. Comparison of CLR normalized TSA counts for indicated proteins under the two tested lysis conditions. Statistical comparisons are Wilcoxon rank sum test with Bonferroni adjusted p-values (ns = not significant; * p adj
    Figure Legend Snippet: ASAP-seq enables a modular and versatile multi-omics toolkit. a. Schematic of experimental design. PBMCs were stained with TBNK panels of the TSA or TSB format at a 1:1 ratio, followed by fixation and permeabilization under mild (LLL) or strong conditions (OMNI). b. Pairwise comparison of centered log-ratio (CLR) normalized TSA and TSB counts for indicated antibodies under mild lysis conditions (n=4,748 cells). Counts were collapsed for unique molecules using UBIs (TSA panel) or UMIs (TSB panel). c. Distribution of percent of mtDNA fragments retained in the library under the two lysis conditions. d. Comparison of CLR normalized TSA counts for indicated proteins under the two tested lysis conditions. Statistical comparisons are Wilcoxon rank sum test with Bonferroni adjusted p-values (ns = not significant; * p adj

    Techniques Used: Staining, Lysis

    ASAP-seq incorporates protein detection in scATAC-seq workflows. a. Schematic of the cell-processing steps that allow retention and profiling of cell-surface markers jointly with chromatin accessibility. Cells are stained with oligo-conjugated antibodies before fixation, permeabilization and transposition with Tn5. b. In droplets, bridge oligos spiked into the barcoding mix promote templated extension of the antibody tags during the first cycle of amplification rendering them complementary to bead-derived barcoding oligos. Extended antibody tags are subsequently barcoded together with the transposed chromatin fragments. c. Species mixing experiment using the Pre-SPRI approach; number of unique nuclear fragments (left) and protein-tag counts (right) associated with each cell barcode. Points are colored based on species classification using ATAC-derived fragments (97.4% agreement by assignment; all but 1 discrepancy was an errant doublet versus singlet classification) d. TSS enrichment scores of mtscATAC-seq without (left) or with concomitant protein tag capture (right). n indicates the number of cells profiled. e. UMAP showing chromatin accessibility-based clustering of PBMCs stained with a 9-antibody panel, with selected markers highlighted. Color bar: protein tag centered log-ratio (CLR) values. f. Cellular distribution of two most commonly detected mtDNA mutations in the population. Thresholds for + were 5% heteroplasmy based on empirical density.
    Figure Legend Snippet: ASAP-seq incorporates protein detection in scATAC-seq workflows. a. Schematic of the cell-processing steps that allow retention and profiling of cell-surface markers jointly with chromatin accessibility. Cells are stained with oligo-conjugated antibodies before fixation, permeabilization and transposition with Tn5. b. In droplets, bridge oligos spiked into the barcoding mix promote templated extension of the antibody tags during the first cycle of amplification rendering them complementary to bead-derived barcoding oligos. Extended antibody tags are subsequently barcoded together with the transposed chromatin fragments. c. Species mixing experiment using the Pre-SPRI approach; number of unique nuclear fragments (left) and protein-tag counts (right) associated with each cell barcode. Points are colored based on species classification using ATAC-derived fragments (97.4% agreement by assignment; all but 1 discrepancy was an errant doublet versus singlet classification) d. TSS enrichment scores of mtscATAC-seq without (left) or with concomitant protein tag capture (right). n indicates the number of cells profiled. e. UMAP showing chromatin accessibility-based clustering of PBMCs stained with a 9-antibody panel, with selected markers highlighted. Color bar: protein tag centered log-ratio (CLR) values. f. Cellular distribution of two most commonly detected mtDNA mutations in the population. Thresholds for + were 5% heteroplasmy based on empirical density.

    Techniques Used: Staining, Amplification, Derivative Assay

    28) Product Images from "Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage"

    Article Title: Correlating cell function and morphology by performing fluorescent immunocytochemical staining on the light-microscope stage

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.180810

    DIC-based corrections of image displacements during the full on-stage ICC. Images were acquired using a CCD camera with high spatial resolution but slow acquisition. Cultured mouse hippocampal neurons were imaged with DIC optics. All panels represent overlays of two images. The DIC image acquired during live-cell imaging (image #1) was used as a spatial reference image and was pseudo-colored red in all panels. DIC images were further acquired during later procedures: immediately after completing chemical fixation (image #2, A ), immediately after membrane permeabilization (image #3, B ), and after completion of ICC procedures (image #4, C ). These test images were pseudo-colored green. In A, B, C , top and bottom rows represent overlays before and after correcting the displacements, respectively. Displacements between two images appear as red or green, and no displacements appear yellow. The degree of displacement, and therefore the degree of required correction, is indicated by how much the test image had to be translated laterally along the x- and y-axes to match the reference image [(Δx, Δy) in pixels]. For a simple demonstration, the illustrated images were selected from the ones acquired without fluorescent filters.
    Figure Legend Snippet: DIC-based corrections of image displacements during the full on-stage ICC. Images were acquired using a CCD camera with high spatial resolution but slow acquisition. Cultured mouse hippocampal neurons were imaged with DIC optics. All panels represent overlays of two images. The DIC image acquired during live-cell imaging (image #1) was used as a spatial reference image and was pseudo-colored red in all panels. DIC images were further acquired during later procedures: immediately after completing chemical fixation (image #2, A ), immediately after membrane permeabilization (image #3, B ), and after completion of ICC procedures (image #4, C ). These test images were pseudo-colored green. In A, B, C , top and bottom rows represent overlays before and after correcting the displacements, respectively. Displacements between two images appear as red or green, and no displacements appear yellow. The degree of displacement, and therefore the degree of required correction, is indicated by how much the test image had to be translated laterally along the x- and y-axes to match the reference image [(Δx, Δy) in pixels]. For a simple demonstration, the illustrated images were selected from the ones acquired without fluorescent filters.

    Techniques Used: Immunocytochemistry, Cell Culture, Live Cell Imaging

    29) Product Images from "Aging and human CD4+ regulatory T cells"

    Article Title: Aging and human CD4+ regulatory T cells

    Journal: Mechanisms of ageing and development

    doi: 10.1016/j.mad.2009.06.003

    Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining
    Figure Legend Snippet: Chemokine receptor expression on CD4 + ,FOXP3 + T cells in young and elderly humans. PBMCs from young (n = 15) and elderly (n = 15) individuals were stained with Abs to CD4, CCR7, CCR4, CCR5, CXCR3 or isotype Abs, followed by permeabilization and staining

    Techniques Used: Expressing, Staining

    30) Product Images from "LipL32 Is a Subsurface Lipoprotein of Leptospirainterrogans: Presentation of New Data and Reevaluation of Previous Studies"

    Article Title: LipL32 Is a Subsurface Lipoprotein of Leptospirainterrogans: Presentation of New Data and Reevaluation of Previous Studies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051025

    Confirmation of subsurface locale of LipL32 by surface IFA and various outer-membrane permeabilization methods. Intact spirochetes or cells disrupted by methanol, vortexing and high-speed centrifugation, 2 mM EDTA or shear force were probed with affinity purified LipL32 antibodies from leptospirosis patient sera or FlaA2 rabbit serum as a control. The data is representation of three experiments performed separately. Binding of antibodies to leptospires were detected either with Alexa Fluor 488 conjugated goat anti-human IgG fragments (for LipL32) or Alexa Fluor 488 conjugated goat anti-rabbit IgG fragments (for FlaA2). A DAPI counterstain was used to demonstrate the presence of spirochetes. The identities of individual proteins recognized by the particular antibody reagent are indicated on the top of each column.
    Figure Legend Snippet: Confirmation of subsurface locale of LipL32 by surface IFA and various outer-membrane permeabilization methods. Intact spirochetes or cells disrupted by methanol, vortexing and high-speed centrifugation, 2 mM EDTA or shear force were probed with affinity purified LipL32 antibodies from leptospirosis patient sera or FlaA2 rabbit serum as a control. The data is representation of three experiments performed separately. Binding of antibodies to leptospires were detected either with Alexa Fluor 488 conjugated goat anti-human IgG fragments (for LipL32) or Alexa Fluor 488 conjugated goat anti-rabbit IgG fragments (for FlaA2). A DAPI counterstain was used to demonstrate the presence of spirochetes. The identities of individual proteins recognized by the particular antibody reagent are indicated on the top of each column.

    Techniques Used: Immunofluorescence, Centrifugation, Affinity Purification, Binding Assay

    31) Product Images from "Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae"

    Article Title: Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00185-13

    NTHi cells demonstrate high intracellular staining after permeabilization by flow cytometric analysis. Only background staining is detected in NTHi cells stained with (no primary) secondary Alexa Fluor 488 (surface staining) (A) or secondary Alexa Fluor
    Figure Legend Snippet: NTHi cells demonstrate high intracellular staining after permeabilization by flow cytometric analysis. Only background staining is detected in NTHi cells stained with (no primary) secondary Alexa Fluor 488 (surface staining) (A) or secondary Alexa Fluor

    Techniques Used: Staining, Flow Cytometry

    32) Product Images from "Distinct Neurotoxicity Profile of Listeriolysin O from Listeria monocytogenes"

    Article Title: Distinct Neurotoxicity Profile of Listeriolysin O from Listeria monocytogenes

    Journal: Toxins

    doi: 10.3390/toxins9010034

    Lytic capacity of listeriolysin O (LLO) in primary glial cells: ( a ) lactate dehydrogenase (LDH) release in primary glial cells after challenge with various concentrations of LLO for 30 min. The red line indicates background LDH release; ( b ) LDH release in acute brain slices, oxygenated with carbogen (95% O 2 /5% CO 2 mix) after 5 h of LLO exposure; ( c ) LDH release in primary glial cultures after challenge with various concentrations of pneumolysin (PLY) for 30 min. 100% lysis controls were prepared by cell lysis with 1% Triton X-100 in PBS; ( d ) live imaging permeabilization (as judged by propidium iodide nuclear staining) analysis in primary mouse glial cultures after challenge with various amounts of LLO and ( e ) PLY. Total number of cells per field was determined by DAPI nuclear staining at the end of the experiment. Values from non-linear regression analysis of half-times are presented in the table. In ( d , e ), toxin concentrations were expressed both as µg/mL and in hemolytic units (HU/mL). All values represent mean ± SEM, n = 4–6 independent experiments; * p
    Figure Legend Snippet: Lytic capacity of listeriolysin O (LLO) in primary glial cells: ( a ) lactate dehydrogenase (LDH) release in primary glial cells after challenge with various concentrations of LLO for 30 min. The red line indicates background LDH release; ( b ) LDH release in acute brain slices, oxygenated with carbogen (95% O 2 /5% CO 2 mix) after 5 h of LLO exposure; ( c ) LDH release in primary glial cultures after challenge with various concentrations of pneumolysin (PLY) for 30 min. 100% lysis controls were prepared by cell lysis with 1% Triton X-100 in PBS; ( d ) live imaging permeabilization (as judged by propidium iodide nuclear staining) analysis in primary mouse glial cultures after challenge with various amounts of LLO and ( e ) PLY. Total number of cells per field was determined by DAPI nuclear staining at the end of the experiment. Values from non-linear regression analysis of half-times are presented in the table. In ( d , e ), toxin concentrations were expressed both as µg/mL and in hemolytic units (HU/mL). All values represent mean ± SEM, n = 4–6 independent experiments; * p

    Techniques Used: Lysis, Imaging, Staining

    33) Product Images from "Uukuniemi Virus as a Tick-Borne Virus Model"

    Article Title: Uukuniemi Virus as a Tick-Borne Virus Model

    Journal: Journal of Virology

    doi: 10.1128/JVI.00095-16

    Characterization of UUKV rescued from plasmids. The UUKV lab strain and rUUKV were analyzed by SDS-PAGE and Western blotting under reducing conditions (A) using the rabbit polyclonal antibody U2 against the three structural viral proteins N, G N , and G C or under nonreducing conditions (B) with the mouse monoclonal antibodies 8B11A3, 6G9E5, and 3D8B3 that recognize each of the structural proteins N, G N , and G C , respectively. (C) BHK-21 cells were exposed to the UUKV lab strain or rUUKV at an MOI of 0.1 for 24 h. After fixation and permeabilization, infected cells were immunostained for N, G N , and G C with the mouse monoclonal antibodies 8B11A3, 6G9E5, and 3D8B3, respectively, and analyzed by flow cytometry. SSC-H, side scatter, height. (D) Infection of BHK-21 cells by UUKV and rUUKV was monitored over 64 h using the flow cytometry-based assay used for the experiment shown in panel C. Infection is given as the percentage of N protein-positive cells. (E) Supernatants collected from cells infected at an MOI of 0.1 and at indicated times were assessed for the production of infectious viral progeny by focus-forming assay.
    Figure Legend Snippet: Characterization of UUKV rescued from plasmids. The UUKV lab strain and rUUKV were analyzed by SDS-PAGE and Western blotting under reducing conditions (A) using the rabbit polyclonal antibody U2 against the three structural viral proteins N, G N , and G C or under nonreducing conditions (B) with the mouse monoclonal antibodies 8B11A3, 6G9E5, and 3D8B3 that recognize each of the structural proteins N, G N , and G C , respectively. (C) BHK-21 cells were exposed to the UUKV lab strain or rUUKV at an MOI of 0.1 for 24 h. After fixation and permeabilization, infected cells were immunostained for N, G N , and G C with the mouse monoclonal antibodies 8B11A3, 6G9E5, and 3D8B3, respectively, and analyzed by flow cytometry. SSC-H, side scatter, height. (D) Infection of BHK-21 cells by UUKV and rUUKV was monitored over 64 h using the flow cytometry-based assay used for the experiment shown in panel C. Infection is given as the percentage of N protein-positive cells. (E) Supernatants collected from cells infected at an MOI of 0.1 and at indicated times were assessed for the production of infectious viral progeny by focus-forming assay.

    Techniques Used: SDS Page, Western Blot, Infection, Flow Cytometry, Cytometry, Focus Forming Assay

    34) Product Images from "Blockade of the PI-3K signaling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro-inflammatory cytokines"

    Article Title: Blockade of the PI-3K signaling pathway by the Aggregatibacter actinomycetemcomitans cytolethal distending toxin induces macrophages to synthesize and secrete pro-inflammatory cytokines

    Journal: Cellular microbiology

    doi: 10.1111/cmi.12299

    Immunofluorescence analysis of internalization of CdtB. THP-1 derived macrophages were exposed to media alone (black dotted line), Cdt WT (red line) or CdtABC Y71P (green line) for 1 hr and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization and staining with anti-CdtB mAb conjugated to AlexaFluor 488. Panel A shows results of cells incubated at 37°C, fixed, permeabilized and stained for CdtB. Panel B shows results of cells incubated at 5°C and treated as described above. Panel C shows fluorescence associated with CdtB in non-permeabilized cells incubated at 37°C. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF); at least 10,000 cells were analyzed per sample. Results are representative of three experiments. The MCF for untreated cells (autofluorescent) was 4.
    Figure Legend Snippet: Immunofluorescence analysis of internalization of CdtB. THP-1 derived macrophages were exposed to media alone (black dotted line), Cdt WT (red line) or CdtABC Y71P (green line) for 1 hr and then analyzed by immunofluorescence and flow cytometry for the presence of CdtB following fixation, permeabilization and staining with anti-CdtB mAb conjugated to AlexaFluor 488. Panel A shows results of cells incubated at 37°C, fixed, permeabilized and stained for CdtB. Panel B shows results of cells incubated at 5°C and treated as described above. Panel C shows fluorescence associated with CdtB in non-permeabilized cells incubated at 37°C. Fluorescence is plotted versus relative cell number. Numbers represent the mean channel fluorescence (MCF); at least 10,000 cells were analyzed per sample. Results are representative of three experiments. The MCF for untreated cells (autofluorescent) was 4.

    Techniques Used: Immunofluorescence, Derivative Assay, Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

    35) Product Images from "Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry"

    Article Title: Analysis of cell surface and intranuclear markers on non-stimulated human PBMC using mass cytometry

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194593

    Effects of different buffers on the detection of rare populations. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). This data compare the frequency of various CD45+ populations between the CS condition and the different permeabilization conditions. a.) Frequencies of rare CD4+ T cell populations: primed T cells (CXCR5+ CCR7+) and Treg cells (CD25 hi CD127 low ). b.) Frequencies of rare B cell populations such as transitional B cells (CD24 hi CD38 hi ) and un-switched memory B cells (CD19+IgD+CD27+). c.) Statistics showing the comparison of the frequency of rare T and B cell populations within the different experimental conditions. The concentrations of antibodies used were: CXCR5 (0.04 mg/ml), CCR7 (0.5 mg/ml), CD25 (0.5 mg/ml), CD127 (0.5 mg/ml), CD24 (0.3 mg/ml), CD38 (0.3 mg/ml), IgD (0.25 mg/ml) and CD27 (0.1 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Effects of different buffers on the detection of rare populations. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). This data compare the frequency of various CD45+ populations between the CS condition and the different permeabilization conditions. a.) Frequencies of rare CD4+ T cell populations: primed T cells (CXCR5+ CCR7+) and Treg cells (CD25 hi CD127 low ). b.) Frequencies of rare B cell populations such as transitional B cells (CD24 hi CD38 hi ) and un-switched memory B cells (CD19+IgD+CD27+). c.) Statistics showing the comparison of the frequency of rare T and B cell populations within the different experimental conditions. The concentrations of antibodies used were: CXCR5 (0.04 mg/ml), CCR7 (0.5 mg/ml), CD25 (0.5 mg/ml), CD127 (0.5 mg/ml), CD24 (0.3 mg/ml), CD38 (0.3 mg/ml), IgD (0.25 mg/ml) and CD27 (0.1 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Staining

    Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Detection of intranuclear markers using the adapted BD cytofix/cytoperm protocol. Cells from healthy donors were incubated with antibodies targeting cell surface antigens and then split into 5 for the following conditions: the “surface staining only” conditions (CS), and fixation and permeabilization using either BD cytofix/cytoperm buffer (ICSb), eBioscience fixation and permeabilization buffer (INSb 1), Maxpar NASB (INSb 2) and PFA/methanol (INSb 3). Next cells were labelled with a mix of antibodies targeting intranuclear markers. Data compare the frequencies of Treg cells (CD25hiFoxP3+), Tfh cells (CD4+BCL6+), Th17 cells (CD4+RoryT+) and CD8+Tbet+ cells between the various permeabilization conditions. The concentrations of antibodies used are as follow: FoxP3 (0.3 mg/ml), BCL6 (0.8 mg/ml), RoryT (0.6 mg/ml) and Tbet (0.3 mg/ml). Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Incubation, Staining

    Visualization of the effects of different buffers on the intensity of cell surface markers. Cells from healthy donors were labeled with antibodies targeting both cell surface antigens and intranuclear antigens. Data show the distribution of CD45+ live cells on viSNE plots with the “cell surface staining” only condition (CS) and the different permeabilization conditions: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). The intensity of CD19+, CD16+, CD56+, CD14+ and HLADR+ events are shown. The concentrations of antibodies used for the detection of these markers are as follows: CD19 (0.5 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). viSNE was performed using 1000 iterations, with a perplexity of 30 and theta = 0.3. The data shown are representative of an independent experiment and represent median with interquartile. Experiments were performed 3 times independently. Different healthy individuals were used for each independent experiment.
    Figure Legend Snippet: Visualization of the effects of different buffers on the intensity of cell surface markers. Cells from healthy donors were labeled with antibodies targeting both cell surface antigens and intranuclear antigens. Data show the distribution of CD45+ live cells on viSNE plots with the “cell surface staining” only condition (CS) and the different permeabilization conditions: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). The intensity of CD19+, CD16+, CD56+, CD14+ and HLADR+ events are shown. The concentrations of antibodies used for the detection of these markers are as follows: CD19 (0.5 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). viSNE was performed using 1000 iterations, with a perplexity of 30 and theta = 0.3. The data shown are representative of an independent experiment and represent median with interquartile. Experiments were performed 3 times independently. Different healthy individuals were used for each independent experiment.

    Techniques Used: Labeling, Staining

    Partial loss of the signal intensity of CD4 and CD127 after barcoding. Cells were thawed as described above and used for the following purposes: a) assessment of the effects of barcoding on the expression of surface markers. Three experimental conditions were performed: no barcode/no permeabilization, surface staining before barcoding and finally barcoding before surface staining. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The concentrations of antibodies used are as follow: CD45 (0.5 mg/ml), CD3 (0.08 mg/ml), CD4 (0.25 mg/ml), CD127 (0.5 mg/ml) and CD25 (0.5 mg/ml). b) Assessment of the cause of the lower signal intensity of CD4 and CD127 when PBMC are barcoded before cell surface staining. 3 conditions were evaluated: no barcode/no permeabilization, permeabilization only/no barcoding and permeabilization followed by barcoding. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The data shown are representative of an independent experiment. Experiments were performed 3 times independently. PBMC from different individuals were used for each independent experiment.
    Figure Legend Snippet: Partial loss of the signal intensity of CD4 and CD127 after barcoding. Cells were thawed as described above and used for the following purposes: a) assessment of the effects of barcoding on the expression of surface markers. Three experimental conditions were performed: no barcode/no permeabilization, surface staining before barcoding and finally barcoding before surface staining. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The concentrations of antibodies used are as follow: CD45 (0.5 mg/ml), CD3 (0.08 mg/ml), CD4 (0.25 mg/ml), CD127 (0.5 mg/ml) and CD25 (0.5 mg/ml). b) Assessment of the cause of the lower signal intensity of CD4 and CD127 when PBMC are barcoded before cell surface staining. 3 conditions were evaluated: no barcode/no permeabilization, permeabilization only/no barcoding and permeabilization followed by barcoding. Data show histogram overlays of CD45, CD3, CD4, CD127 and CD25 for the different conditions. The data shown are representative of an independent experiment. Experiments were performed 3 times independently. PBMC from different individuals were used for each independent experiment.

    Techniques Used: Expressing, Staining

    Effects of different buffers on the detection of cell surface markers. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). Here, we show the effects of different permeabilization conditions on the frequency of various cell surface markers. a.) Histograms showing the frequency and distribution of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events in the CS condition. The concentrations of antibodies used were: CD45 (0.5 mg/ml), CD19 (0.5 mg/ml), CD3 (0.08 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). b.) Comparison of the frequency of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events between the CS condition and the different permeabilization conditions. Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p
    Figure Legend Snippet: Effects of different buffers on the detection of cell surface markers. Cells from healthy donors were labelled with antibodies targeting both cell surface and intranuclear antigens. Different permeabilization conditions are compared to the cell surface staining only” (CS) condition: ICSb (BD cytofix/cytoperm buffer), INSb 1 (eBioscience permeabilization buffer), INSb 2 (Maxpar NASB) and INSb 3 (Methanol/PFA). Here, we show the effects of different permeabilization conditions on the frequency of various cell surface markers. a.) Histograms showing the frequency and distribution of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events in the CS condition. The concentrations of antibodies used were: CD45 (0.5 mg/ml), CD19 (0.5 mg/ml), CD3 (0.08 mg/ml), CD16 (0.2 mg/ml), CD56 (0.1 mg/ml), CD14 (0.3 mg/ml) and HLADR (0.3 mg/ml). b.) Comparison of the frequency of CD45+, CD19+, CD3+, CD16+, CD56+, CD14+ and HLADR+ events between the CS condition and the different permeabilization conditions. Statistics was performed using one-way ANOVA with Bonferroni’s multiple test correction (*p

    Techniques Used: Staining

    36) Product Images from "Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5"

    Article Title: Induction of Cell Death in the Human Acute Lymphoblastic Leukemia Cell Line Reh by Infection with Rotavirus Isolate Wt1-5

    Journal: Biomedicines

    doi: 10.3390/biomedicines8080242

    Inhibition of Wt1-5 infection after pre-treatment of Reh cells with antibodies to HSPs, Hsc70, PDI and integrin β3. Reh cells and PBMCs were treated with antibodies to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 and labeled with secondary antibodies labeled with FITC, and then, subjected to flow cytometric analysis. ( A ) Flow cytometry analysis for each cellular protein of Reh cells and PBMCs is shown. ( B ) Median fluorescence intensity (MFI) for each cellular protein of Reh cells and PBMCs is shown. Isotype antibodies were used to adjust quadrants. Results are from three different assays. ( C ) Reh cells at a logarithmic growth phase were pre-treated with antibodies (4 or 0.4 mg/mL) to cellular proteins Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 for 1 h at 37 °C. After removal of antibodies, cells were infected with trypsin-activated Wt1-5 at MOI 2 and harvested at 24 h.p.i. before PFD fixation and permeabilization. Viral structural antigens were analyzed by flow cytometry for each antibody treatment using control isotype antibodies to adjust quadrants. Percentages of Wt1-5-infected cells are shown. ( D ) Median fluorescence intensity (MFI) quantification in terms of arbitrary units of fluorescence (AUF) is shown for viral structural proteins for each antibody treatment described in ( C ). ( E ) Cell viability was assessed with the resazurin reduction test for cells that had previously been treated with antibodies to each cellular protein (Hsp90, 7Hsp0, Hsp60, Hsp40, Hsc70, PDI and integrin β3). Wt1-5 infected cells and non-infected and non-antibody-treated cells were used as a control at 24 h.p.i. ( F ) Cell viability of an aliquot of cells assayed in ( E ) but determined with the trypan blue exclusion test is shown. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).
    Figure Legend Snippet: Inhibition of Wt1-5 infection after pre-treatment of Reh cells with antibodies to HSPs, Hsc70, PDI and integrin β3. Reh cells and PBMCs were treated with antibodies to Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 and labeled with secondary antibodies labeled with FITC, and then, subjected to flow cytometric analysis. ( A ) Flow cytometry analysis for each cellular protein of Reh cells and PBMCs is shown. ( B ) Median fluorescence intensity (MFI) for each cellular protein of Reh cells and PBMCs is shown. Isotype antibodies were used to adjust quadrants. Results are from three different assays. ( C ) Reh cells at a logarithmic growth phase were pre-treated with antibodies (4 or 0.4 mg/mL) to cellular proteins Hsp90, Hsp70, Hsp60, Hsp40, Hsc70, PDI and integrin β3 for 1 h at 37 °C. After removal of antibodies, cells were infected with trypsin-activated Wt1-5 at MOI 2 and harvested at 24 h.p.i. before PFD fixation and permeabilization. Viral structural antigens were analyzed by flow cytometry for each antibody treatment using control isotype antibodies to adjust quadrants. Percentages of Wt1-5-infected cells are shown. ( D ) Median fluorescence intensity (MFI) quantification in terms of arbitrary units of fluorescence (AUF) is shown for viral structural proteins for each antibody treatment described in ( C ). ( E ) Cell viability was assessed with the resazurin reduction test for cells that had previously been treated with antibodies to each cellular protein (Hsp90, 7Hsp0, Hsp60, Hsp40, Hsc70, PDI and integrin β3). Wt1-5 infected cells and non-infected and non-antibody-treated cells were used as a control at 24 h.p.i. ( F ) Cell viability of an aliquot of cells assayed in ( E ) but determined with the trypan blue exclusion test is shown. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).

    Techniques Used: Inhibition, Infection, Labeling, Flow Cytometry, Fluorescence

    Nuclear fragmentation induced by Wt1-5 infection of Reh cells. Reh cells at a logarithmic growth phase were infected or not infected with trypsin-activated Wt1-5 (MOI 0.5 to 6) in RPMI culture medium without FBS. UV-inactivated Wt1-5-infected cells and DNAse I-treated cells after permeabilization were used as a control. ( A ) Representative photographs of immunofluorescence analysis for TUNEL assays are shown. Cells were labeled with Alexa Fluor 488 (green) for TUNEL, Alexa Fluor 568 (red) for Wt1-5 structural proteins and DAPI (blue) for nuclei. ( B ) Quantitative analysis of images shown in (A) is presented as percentages of cells being positive to TUNEL at the indicated MOIs at 24 h.p.i. (+/+): positive TUNEL cells that are also positive for Wt1-5 structural proteins; (−/−): cells negative to both TUNEL and Wt1-5 structural proteins. ( C ) DNA fragmentation pattern from Wt1-5-infected cells at MOI 1 and 6 is shown in an agarose gel (1%) after staining with SyBR ® Safe DNA gel stain at 12 and 24 h.p.i. ( D ) Representative photographs of immunofluorescence assays for PARP-1 cleavage at 24 h.p.i. are shown. Cleaved PARP-1 was labeled with FITC (green), Wt1-5 structural proteins with Alexa Fluor 568 (red) and nuclei with DAPI (blue). ( E ) Quantitative analysis of images presented in (d) is shown as percentages of cleaved PARP-1-positive cells. (+/+): positive cells to both cleaved PARP-1 and Wt1-5 structural proteins; (−/−): cells negative to both cleaved PARP-1 and Wt1-5 structural proteins. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).
    Figure Legend Snippet: Nuclear fragmentation induced by Wt1-5 infection of Reh cells. Reh cells at a logarithmic growth phase were infected or not infected with trypsin-activated Wt1-5 (MOI 0.5 to 6) in RPMI culture medium without FBS. UV-inactivated Wt1-5-infected cells and DNAse I-treated cells after permeabilization were used as a control. ( A ) Representative photographs of immunofluorescence analysis for TUNEL assays are shown. Cells were labeled with Alexa Fluor 488 (green) for TUNEL, Alexa Fluor 568 (red) for Wt1-5 structural proteins and DAPI (blue) for nuclei. ( B ) Quantitative analysis of images shown in (A) is presented as percentages of cells being positive to TUNEL at the indicated MOIs at 24 h.p.i. (+/+): positive TUNEL cells that are also positive for Wt1-5 structural proteins; (−/−): cells negative to both TUNEL and Wt1-5 structural proteins. ( C ) DNA fragmentation pattern from Wt1-5-infected cells at MOI 1 and 6 is shown in an agarose gel (1%) after staining with SyBR ® Safe DNA gel stain at 12 and 24 h.p.i. ( D ) Representative photographs of immunofluorescence assays for PARP-1 cleavage at 24 h.p.i. are shown. Cleaved PARP-1 was labeled with FITC (green), Wt1-5 structural proteins with Alexa Fluor 568 (red) and nuclei with DAPI (blue). ( E ) Quantitative analysis of images presented in (d) is shown as percentages of cleaved PARP-1-positive cells. (+/+): positive cells to both cleaved PARP-1 and Wt1-5 structural proteins; (−/−): cells negative to both cleaved PARP-1 and Wt1-5 structural proteins. Data are shown as mean ± SD of three independent experiments performed in duplicate. Statistical significance is indicated by p -values (*** p ≤ 0.01, ** p ≤ 0.05, and * p ≤ 0.1).

    Techniques Used: Infection, Immunofluorescence, TUNEL Assay, Labeling, Agarose Gel Electrophoresis, Staining

    37) Product Images from "Infection of human lymphomononuclear cells by SARS-CoV-2"

    Article Title: Infection of human lymphomononuclear cells by SARS-CoV-2

    Journal: bioRxiv

    doi: 10.1101/2020.07.28.225912

    Flow cytometry (FC) of SARS-CoV-2-infected PBMCs from healthy with labeling for SARS-CoV-2. PMBCs from healthy donors infected in vitro (MOI=1) were analyzed by FC using mouse polyclonal anti-SARS-CoV-2 with and without cell permeabilization. Treatment with trypsin to remove surface-bound viral particles was used as an additional control. (A) Representative histograms of surface and intracellular staining for SARS-CoV-2, with SARS-CoV-2-infected cells in red and trypsin-treated infected cells in black. (B) Comparison of intracellular and surface staining of infected cells treated or not with trypsin, and non-infected cells in percentages on the left and MFI on the right.
    Figure Legend Snippet: Flow cytometry (FC) of SARS-CoV-2-infected PBMCs from healthy with labeling for SARS-CoV-2. PMBCs from healthy donors infected in vitro (MOI=1) were analyzed by FC using mouse polyclonal anti-SARS-CoV-2 with and without cell permeabilization. Treatment with trypsin to remove surface-bound viral particles was used as an additional control. (A) Representative histograms of surface and intracellular staining for SARS-CoV-2, with SARS-CoV-2-infected cells in red and trypsin-treated infected cells in black. (B) Comparison of intracellular and surface staining of infected cells treated or not with trypsin, and non-infected cells in percentages on the left and MFI on the right.

    Techniques Used: Flow Cytometry, Infection, Labeling, In Vitro, Staining

    38) Product Images from "A hidden gene in astroviruses encodes a viroporin"

    Article Title: A hidden gene in astroviruses encodes a viroporin

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17906-x

    XPs from different astroviruses have viroporin-like activity. a Schematic representation of SINV replicon C used to evaluate cell-permeabilizing activity of expressed proteins. SINV elements: nsP1-4 – non-structural polyprotein; CP – capsid protein; SG – subgenomic promoter. b Membrane permeabilization in BSR cells at 8 h post RNA electroporation with Sindbis virus replicons (SINV repC) expressing HAstV1 XP, enterovirus Strep-2B (positive control), or mCherry (negative control). Ongoing protein synthesis was labeled with 1 mM AHA in the presence or absence of 1 mM HB as a translation inhibitor. Cells were lysed and AHA-bearing proteins were ligated to the fluorescent reporter IRDye800CW Alkyne by click chemistry, separated by SDS-PAGE, and visualized by in-gel fluorescence. Numbers below each pair of samples indicate protein synthesis quantified for HB-treated cells relative to values obtained for untreated cells. c Statistical analysis of membrane permeabilization caused by different XPs and mutants and the indicated control proteins in BSR cells. Bars indicate the amount of protein synthesis in HB-treated cells relative to untreated cells. P values are from comparisons with mock (BSR; black) or with pSINV-repC-XP (blue). d Sequences of wt, RR1, and RR2 HAstV1 mutants. e Sequences of HAstV1, HAstV4, and feline (FAstV), canine (CAstV) and porcine (PAstV) astrovirus XPs. f Alignment of pAVIC1-wt and -5L showing translation of XP (red) and the overlapping CP (blue). Introduced nucleotide and amino acid changes are highlighted in yellow. g Quantification of virus titer, RNA, and protein levels in released virions and infected cells as described in Fig. 4g . h Caco2 cells were infected with the indicated viruses at MOI 0.2 and incubated for 48 h. Released virus in clarified supernatants was titrated. i Caco2 cells were infected with the indicated viruses at MOI 0.2 in the presence (dark blue) or absence (light blue) of 5 µM hexamethylene amiloride (HMA). Intracellular (HMA-free) virus was titrated. See Supplementary Fig. 20 for cell toxicity data. P values come from two-tailed t -tests without adjustment for multiple comparisons. Error bars indicate mean ± s.d.; n = 3 ( c , g , h ) or 4 ( i ) biologically independent experiments. Source data are provided as a Source Data file.
    Figure Legend Snippet: XPs from different astroviruses have viroporin-like activity. a Schematic representation of SINV replicon C used to evaluate cell-permeabilizing activity of expressed proteins. SINV elements: nsP1-4 – non-structural polyprotein; CP – capsid protein; SG – subgenomic promoter. b Membrane permeabilization in BSR cells at 8 h post RNA electroporation with Sindbis virus replicons (SINV repC) expressing HAstV1 XP, enterovirus Strep-2B (positive control), or mCherry (negative control). Ongoing protein synthesis was labeled with 1 mM AHA in the presence or absence of 1 mM HB as a translation inhibitor. Cells were lysed and AHA-bearing proteins were ligated to the fluorescent reporter IRDye800CW Alkyne by click chemistry, separated by SDS-PAGE, and visualized by in-gel fluorescence. Numbers below each pair of samples indicate protein synthesis quantified for HB-treated cells relative to values obtained for untreated cells. c Statistical analysis of membrane permeabilization caused by different XPs and mutants and the indicated control proteins in BSR cells. Bars indicate the amount of protein synthesis in HB-treated cells relative to untreated cells. P values are from comparisons with mock (BSR; black) or with pSINV-repC-XP (blue). d Sequences of wt, RR1, and RR2 HAstV1 mutants. e Sequences of HAstV1, HAstV4, and feline (FAstV), canine (CAstV) and porcine (PAstV) astrovirus XPs. f Alignment of pAVIC1-wt and -5L showing translation of XP (red) and the overlapping CP (blue). Introduced nucleotide and amino acid changes are highlighted in yellow. g Quantification of virus titer, RNA, and protein levels in released virions and infected cells as described in Fig. 4g . h Caco2 cells were infected with the indicated viruses at MOI 0.2 and incubated for 48 h. Released virus in clarified supernatants was titrated. i Caco2 cells were infected with the indicated viruses at MOI 0.2 in the presence (dark blue) or absence (light blue) of 5 µM hexamethylene amiloride (HMA). Intracellular (HMA-free) virus was titrated. See Supplementary Fig. 20 for cell toxicity data. P values come from two-tailed t -tests without adjustment for multiple comparisons. Error bars indicate mean ± s.d.; n = 3 ( c , g , h ) or 4 ( i ) biologically independent experiments. Source data are provided as a Source Data file.

    Techniques Used: Activity Assay, Electroporation, Expressing, Positive Control, Negative Control, Labeling, SDS Page, Fluorescence, Infection, Incubation, Two Tailed Test

    39) Product Images from "STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells"

    Article Title: STIM1‐mediated calcium influx controls antifungal immunity and the metabolic function of non‐pathogenic Th17 cells

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201911592

    STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P
    Figure Legend Snippet: STIM 1 p.L374P mutation causes defect in T‐cell proliferation and cytokine production Cell size (A) and proliferation (B) of CD4 + T cells from P1 (red), P2 (blue), their mother (gray), and a HD (black) stimulated with anti‐CD3 (5 μg/ml) and anti‐CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 h. (A) Representative histograms of FSC (left panel) and percentages of T‐cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments. Cytokine production by PBMC from P1, P2, the mother, and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 h. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4, and CD45RO, permeabilization and intracellular cytokine staining for GM‐CSF, IL‐22, and IL‐17A. Representative flow cytometry plots (C) and quantification of Th17 (GM‐CSF, IL‐22, IL‐17A), Th1 (TNF‐α, IFN‐γ), and Th2 (IL‐4) cytokines (D). Data represent the mean ± SEM from two independent experiments. Data information: Statistical analysis by unpaired Student's t ‐test. * P

    Techniques Used: Mutagenesis, Flow Cytometry, Staining

    40) Product Images from "Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection"

    Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02920

    Molecular details of the M2e-VHH-23m: M2e interaction . (A) Crystal structure of M2e-VHH-23m in complex with the M2e peptide, showing the linear binding epitope of M2e residues 6–13 binding to a shallow groove on the surface of M2e-VHH-23m (Interface 1). Left: The M2e peptide is shown in cyan stick representation, the M2e-VHH-23m in green cartoon representation. CDR2 and CDR3 are colored pale and lime green, respectively. Right: Details of the interactions between M2e residues 6–13 and the residues making up interface 1 on M2e-VHH-23m. (B) Details of the interactions between M2e residues 14–19 and the residues making up interface 2 on M2e-VHH-23m. Coloring of M2e-VHH-23m as in A, M2e shown in gray stick representation. (C) Docking of the M2e peptide on the crystal structure of M2e-VHH-23m confirms the ligand swapping hypothesis, showing that M2e binds an extended grove on M2e-VHH-23m comprising both Interface 1 and Interface 2. Coloring of M2e-VHH-23m as in A, M2e shown in yellow stick representation. (D) Binding of M2e-VHH-23m to M2e Ala scan mutants. HEK293T cells were transfected with Flag-tagged M2 wild type (WT) and M2e Ala scan mutant expression constructs and subsequently incubated with 20 μg/ml of M2e-VHH-23m or F-VHH-4. After fixation with 2% paraformaldehyde and permeabilization, binding was detected with a mouse anti-Histidine tag antibody and rabbit anti-Fag tag antibody followed by an anti-mouse IgG Alexa 647 and anti-rabbit IgG Alexa 488, respectively. The median fluorescence intensity (MFI) was calculated by subtracting the median fluorescence of binding of M2e-VHH-23m or F-VHH-4 to transfected cells from the median fluorescence of non-transfected cells bound by M2e-VHH-23m or F-VHH-4.
    Figure Legend Snippet: Molecular details of the M2e-VHH-23m: M2e interaction . (A) Crystal structure of M2e-VHH-23m in complex with the M2e peptide, showing the linear binding epitope of M2e residues 6–13 binding to a shallow groove on the surface of M2e-VHH-23m (Interface 1). Left: The M2e peptide is shown in cyan stick representation, the M2e-VHH-23m in green cartoon representation. CDR2 and CDR3 are colored pale and lime green, respectively. Right: Details of the interactions between M2e residues 6–13 and the residues making up interface 1 on M2e-VHH-23m. (B) Details of the interactions between M2e residues 14–19 and the residues making up interface 2 on M2e-VHH-23m. Coloring of M2e-VHH-23m as in A, M2e shown in gray stick representation. (C) Docking of the M2e peptide on the crystal structure of M2e-VHH-23m confirms the ligand swapping hypothesis, showing that M2e binds an extended grove on M2e-VHH-23m comprising both Interface 1 and Interface 2. Coloring of M2e-VHH-23m as in A, M2e shown in yellow stick representation. (D) Binding of M2e-VHH-23m to M2e Ala scan mutants. HEK293T cells were transfected with Flag-tagged M2 wild type (WT) and M2e Ala scan mutant expression constructs and subsequently incubated with 20 μg/ml of M2e-VHH-23m or F-VHH-4. After fixation with 2% paraformaldehyde and permeabilization, binding was detected with a mouse anti-Histidine tag antibody and rabbit anti-Fag tag antibody followed by an anti-mouse IgG Alexa 647 and anti-rabbit IgG Alexa 488, respectively. The median fluorescence intensity (MFI) was calculated by subtracting the median fluorescence of binding of M2e-VHH-23m or F-VHH-4 to transfected cells from the median fluorescence of non-transfected cells bound by M2e-VHH-23m or F-VHH-4.

    Techniques Used: Binding Assay, Transfection, Mutagenesis, Expressing, Construct, Incubation, Fluorescence

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    Article Title: The Extracellular Matrix Influences Ovarian Carcinoma Cells’ Sensitivity to Cisplatinum: A First Step towards Personalized Medicine
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    other:

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