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Becton Dickinson facs canto ii flow cytometer
Flow <t>cytometry</t> pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting <t>(FACS)</t> of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.
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1) Product Images from "Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris"

Article Title: Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Journal: PLoS ONE

doi: 10.1371/journal.pone.0097269

Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.
Figure Legend Snippet: Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

Techniques Used: Flow Cytometry, Cytometry, Fluorescence, FACS, Marker, Software, Standard Deviation

2) Product Images from "Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes"

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00525

Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p
Figure Legend Snippet: Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

Techniques Used: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p
Figure Legend Snippet: Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

Techniques Used: Transgenic Assay, Mouse Assay, Injection, Negative Control, Staining, FACS

Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p
Figure Legend Snippet: Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p

Techniques Used: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).
Figure Legend Snippet: Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

Techniques Used: Activity Assay, Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

3) Product Images from "Platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation"

Article Title: Platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation

Journal: bioRxiv

doi: 10.1101/2020.01.06.895722

Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 – filtered HBS buffer without MPs (negative control); A2, B2 – MPs from erythrocytes; A3, B3 – MPs from platelets; A4, B4 – MPs from THP-1 cells; A5, B5 – MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) (gate P3 in B1-B5) were counted in the SSC/FSC window (A1-A5) in the size gate
Figure Legend Snippet: Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 – filtered HBS buffer without MPs (negative control); A2, B2 – MPs from erythrocytes; A3, B3 – MPs from platelets; A4, B4 – MPs from THP-1 cells; A5, B5 – MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) (gate P3 in B1-B5) were counted in the SSC/FSC window (A1-A5) in the size gate

Techniques Used: Flow Cytometry, FACS, Fluorescence, Negative Control

4) Product Images from "CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities"

Article Title: CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076979

Comparison of the immunomodulatory capacity of CD105 - and CD105 + mASCs. (A) The levels of PGE 2 and TGF-β1 were measured in supernatants from ASC tot , CD105 - and CD105 + mASCs using specific ELISAs (see materials and methods). (B) ASC tot , CD105 - and CD105 + mASCs were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR. (C) Increasing numbers of mitomycin C-treated ASC tot , CD105 - and CD105 + mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 µg/ml) for 3 days. The cells were harvested and acquired on a FACS Canto II flow cytometer and the proliferation of CD4 + splenocytes was quantified using the FlowJo software. (D) BM-MΦs were cultured with or without mASC for 48 hours and then restimulated with LPS (1 µg/ml) for 24 hours. The levels of IL-10 (left graph) and IL-12 (right graph) in the supernatants of the co-cultures were measured using specific ELISAs. Data is shown as mean (SEM) of at least 3 independent experiments. *=p
Figure Legend Snippet: Comparison of the immunomodulatory capacity of CD105 - and CD105 + mASCs. (A) The levels of PGE 2 and TGF-β1 were measured in supernatants from ASC tot , CD105 - and CD105 + mASCs using specific ELISAs (see materials and methods). (B) ASC tot , CD105 - and CD105 + mASCs were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR. (C) Increasing numbers of mitomycin C-treated ASC tot , CD105 - and CD105 + mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 µg/ml) for 3 days. The cells were harvested and acquired on a FACS Canto II flow cytometer and the proliferation of CD4 + splenocytes was quantified using the FlowJo software. (D) BM-MΦs were cultured with or without mASC for 48 hours and then restimulated with LPS (1 µg/ml) for 24 hours. The levels of IL-10 (left graph) and IL-12 (right graph) in the supernatants of the co-cultures were measured using specific ELISAs. Data is shown as mean (SEM) of at least 3 independent experiments. *=p

Techniques Used: Purification, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Labeling, FACS, Flow Cytometry, Cytometry, Software

5) Product Images from "Cul4 E3 ubiquitin ligase regulates ovarian cancer drug resistance by targeting the antiapoptotic protein BIRC3"

Article Title: Cul4 E3 ubiquitin ligase regulates ovarian cancer drug resistance by targeting the antiapoptotic protein BIRC3

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1200-y

CRL4 knockdown increases the apoptosis of A2780CP cells in response to cisplatin. a Flow cytometry analysis showing apoptosis of A2780CP cells transduced with virus containing scrambled shRNA (NT) or shRNA against DDB1 or Cul4A. Quantification data were averaged from three independent experiments. Apoptosis rate was the sum of the two quadrats on the right in each FACS figure. Data represent mean ± SD. Significance of differences was calculated using Student’s t test (* P
Figure Legend Snippet: CRL4 knockdown increases the apoptosis of A2780CP cells in response to cisplatin. a Flow cytometry analysis showing apoptosis of A2780CP cells transduced with virus containing scrambled shRNA (NT) or shRNA against DDB1 or Cul4A. Quantification data were averaged from three independent experiments. Apoptosis rate was the sum of the two quadrats on the right in each FACS figure. Data represent mean ± SD. Significance of differences was calculated using Student’s t test (* P

Techniques Used: Flow Cytometry, Cytometry, Transduction, shRNA, FACS

6) Product Images from "Inhibition of Elastase-Pulmonary Emphysema in Dominant-Negative MafB Transgenic Mice"

Article Title: Inhibition of Elastase-Pulmonary Emphysema in Dominant-Negative MafB Transgenic Mice

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.8737

Flow cytometric analysis of AM surface markers. AM cell surface markers (F4/80 and CD11b) were analysed using flow cytometry. BAL cells 7 d after PPE administration were stained with FITC-labelled F4/80 antibody, PerCP-by5.5-labelled CD11b antibody, and PE-labelled Gr-1 antibody and subjected to flow cytometry using the FACS Canto II flow cytometer. To avoid the contamination of neutrophils, Gr-1-positive cells were omitted from the analysis (a). Next, the analysis of Gr-1 negative cells was performed based on their F4/80 and CD11b fluorescence intensity (a). Representative FACS images for WT (b, left) and Tg (b, right) mice are shown. The Gr-1 negative cells were divided into three groups: F4/80-weakly positive (F4/80 low ) and CD11b-intermediately positive (CD11b med ) cells, purple dots; F4/80 med positive and CD11b-highly positive (CD11b high ) cells, green dots; and F4/80 med positive and CD11b-weakly or -intermediately (CD11b low/med ) positive cells, blue dots (c). The ratio of each groups was examined (d). The number of F4/80 med and CD11b low/med cells decreased significantly in the AMs of DN-MafB Tg mice compared with those of WT mice. n = 3 for each group. *: P
Figure Legend Snippet: Flow cytometric analysis of AM surface markers. AM cell surface markers (F4/80 and CD11b) were analysed using flow cytometry. BAL cells 7 d after PPE administration were stained with FITC-labelled F4/80 antibody, PerCP-by5.5-labelled CD11b antibody, and PE-labelled Gr-1 antibody and subjected to flow cytometry using the FACS Canto II flow cytometer. To avoid the contamination of neutrophils, Gr-1-positive cells were omitted from the analysis (a). Next, the analysis of Gr-1 negative cells was performed based on their F4/80 and CD11b fluorescence intensity (a). Representative FACS images for WT (b, left) and Tg (b, right) mice are shown. The Gr-1 negative cells were divided into three groups: F4/80-weakly positive (F4/80 low ) and CD11b-intermediately positive (CD11b med ) cells, purple dots; F4/80 med positive and CD11b-highly positive (CD11b high ) cells, green dots; and F4/80 med positive and CD11b-weakly or -intermediately (CD11b low/med ) positive cells, blue dots (c). The ratio of each groups was examined (d). The number of F4/80 med and CD11b low/med cells decreased significantly in the AMs of DN-MafB Tg mice compared with those of WT mice. n = 3 for each group. *: P

Techniques Used: Flow Cytometry, Cytometry, Staining, FACS, Fluorescence, Mouse Assay, Affinity Magnetic Separation

7) Product Images from "Evaluation of Lapatinib Powder-Entrapped Biodegradable Polymeric Microstructures Fabricated by X-Ray Lithography for a Targeted and Sustained Drug Delivery System"

Article Title: Evaluation of Lapatinib Powder-Entrapped Biodegradable Polymeric Microstructures Fabricated by X-Ray Lithography for a Targeted and Sustained Drug Delivery System

Journal: Materials

doi: 10.3390/ma8020519

Effects of lapatinib and lapatinib powder-entrapped microstructures on gastric cancer cell lines. ( A ) Cells were plated at 3000 cells/well in a 96-well plate. After 24 h, cells were treated with lapatinib at various concentrations (0.01–10 μM). After 72 h, an MTT assay was performed to evaluate the growth inhibitory activity. The absorbance of each well was measured at 540 nm. The percentage of growth is expressed as a percentage of the control (no drug) cell growth; ( B ) NCI-N87 was treated with lapatinib-entrapped microstructures (150, 1500 and 3000 units per 3 mL of RPMI-1640 medium) for 72 h. After the treatment schedule, cells were analyzed for DNA content by flow cytometry. Proportions of the cells in the subG1, G0/G1, S and G2/M phases were quantified with the FACS Diva program; Control (subG1: 1.1%, G0/G1: 62.6%, S: 19.5%, G2/M: 16.8%), 150 units (subG1: 13.1%, G0/G1: 57.2%, S: 17.5%, G2/M: 12.2%), 1500 units (subG1: 20.9%, G0/G1: 58.3%, S: 10.8%, G2/M: 10.0%) and 3000 units (subG1: 27.4%, G0/G1: 51.8%, S: 12.2%, G2/M: 8.6%); ( C ) Cell cycle phases displayed as a histogram.
Figure Legend Snippet: Effects of lapatinib and lapatinib powder-entrapped microstructures on gastric cancer cell lines. ( A ) Cells were plated at 3000 cells/well in a 96-well plate. After 24 h, cells were treated with lapatinib at various concentrations (0.01–10 μM). After 72 h, an MTT assay was performed to evaluate the growth inhibitory activity. The absorbance of each well was measured at 540 nm. The percentage of growth is expressed as a percentage of the control (no drug) cell growth; ( B ) NCI-N87 was treated with lapatinib-entrapped microstructures (150, 1500 and 3000 units per 3 mL of RPMI-1640 medium) for 72 h. After the treatment schedule, cells were analyzed for DNA content by flow cytometry. Proportions of the cells in the subG1, G0/G1, S and G2/M phases were quantified with the FACS Diva program; Control (subG1: 1.1%, G0/G1: 62.6%, S: 19.5%, G2/M: 16.8%), 150 units (subG1: 13.1%, G0/G1: 57.2%, S: 17.5%, G2/M: 12.2%), 1500 units (subG1: 20.9%, G0/G1: 58.3%, S: 10.8%, G2/M: 10.0%) and 3000 units (subG1: 27.4%, G0/G1: 51.8%, S: 12.2%, G2/M: 8.6%); ( C ) Cell cycle phases displayed as a histogram.

Techniques Used: MTT Assay, Activity Assay, Flow Cytometry, Cytometry, FACS

8) Product Images from "CMV promoter mutants with a reduced propensity to productivity loss in CHO cells"

Article Title: CMV promoter mutants with a reduced propensity to productivity loss in CHO cells

Journal: Scientific Reports

doi: 10.1038/srep16952

( a ) hCMV-MIE sequence (left) and list of mutants (right). Positions where C to G mutations were introduced are marked with an asterisk. Transcription factor binding sites are underlined. CpG sites are highlighted in grey. hCMV-MIE variants are coded with a three letter identifier referring to the nucleotides at positions −508, −179 and −41. DNA positions are numbered relative to the transcription start site. (b) Transient expression of SEAP driven by hCMV-MIE mutants. Expression levels were normalised to unmutated hCMV-MIE (CCC). Error bars represent SD of eight biological replicates. Two independent experiments were performed. Potential effects of the hCMV-MIE mutations within the same experiment were tested with Tukey HSD (α = 0.05). Significant differences between the promoter mutants and CCC within the same experiment are marked with the respective p-value. (c) eGFP expression of permanently transfected CHO cell pools 34 days (upper panel) and 87 days (lower panel) after transfection. eGFP expression was quantified by FACS and the geometrical mean of the fluorescence intensity (gMFI) was plotted for each cell pool. The identity of the promoter variants and the number (N) of independent cell pools is indicated below the x-axis. The upper and the lower ends of the boxes represent the first and the third quartile of each group. The ends of the whiskers represent the lowest and highest values still within the 1.5fold interquartile range. The grey line indicates the overall mean of the gMFI values. (d) eGFP expression of permanently transfected CHO cell pools 42 days (left panel) and 69 days (right panel) after transfection; all further labelling is identical to ( c ). (e) Percentage methylation levels of hCMV-MIE variants at each CpG cytosine 42 days (upper panels) and 69 days (lower panels) after transfection. Methylation levels are plotted against DNA positions numbered 5′ to 3′. The positions of C-508, C-179, and C-41 are highlighted.
Figure Legend Snippet: ( a ) hCMV-MIE sequence (left) and list of mutants (right). Positions where C to G mutations were introduced are marked with an asterisk. Transcription factor binding sites are underlined. CpG sites are highlighted in grey. hCMV-MIE variants are coded with a three letter identifier referring to the nucleotides at positions −508, −179 and −41. DNA positions are numbered relative to the transcription start site. (b) Transient expression of SEAP driven by hCMV-MIE mutants. Expression levels were normalised to unmutated hCMV-MIE (CCC). Error bars represent SD of eight biological replicates. Two independent experiments were performed. Potential effects of the hCMV-MIE mutations within the same experiment were tested with Tukey HSD (α = 0.05). Significant differences between the promoter mutants and CCC within the same experiment are marked with the respective p-value. (c) eGFP expression of permanently transfected CHO cell pools 34 days (upper panel) and 87 days (lower panel) after transfection. eGFP expression was quantified by FACS and the geometrical mean of the fluorescence intensity (gMFI) was plotted for each cell pool. The identity of the promoter variants and the number (N) of independent cell pools is indicated below the x-axis. The upper and the lower ends of the boxes represent the first and the third quartile of each group. The ends of the whiskers represent the lowest and highest values still within the 1.5fold interquartile range. The grey line indicates the overall mean of the gMFI values. (d) eGFP expression of permanently transfected CHO cell pools 42 days (left panel) and 69 days (right panel) after transfection; all further labelling is identical to ( c ). (e) Percentage methylation levels of hCMV-MIE variants at each CpG cytosine 42 days (upper panels) and 69 days (lower panels) after transfection. Methylation levels are plotted against DNA positions numbered 5′ to 3′. The positions of C-508, C-179, and C-41 are highlighted.

Techniques Used: Sequencing, Binding Assay, Expressing, Countercurrent Chromatography, Transfection, FACS, Fluorescence, Methylation

9) Product Images from "NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation"

Article Title: NOX1-induced accumulation of reactive oxygen species in abdominal fat-derived mesenchymal stromal cells impinges on long-term proliferation

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.84

A specific NOX1 inhibitor reduces ROS accumulation and promotes long-term expansion of aASCs. Abdominal ASCs (Abd) were propagated under normoxic oxygen conditions (21% oxygen) in the presence of an NOX1-specific inhibitor ML171 (0, 0.5, 2.5 and 5 μ M). The following analyses were performed at the indicated passages (P). ( a I) ROS accumulation was evaluated by FACS (fluorescence-activated cell sorting) analysis using DCFDA staining, and the results of untreated aASCs compared with aASCs treated with 5 μ M inhibitor are shown. ( a II) Starting from P3, an additional population with a low DCFDA signal appeared (marked by an arrow). Dual staining of cells by DCFDA and aqua blue dye (an amine viability dye) was performed and analyzed by FACS. Results demonstrated that the majority of low DCFDA cells were aqua blue-positive dead cells. ( a III) RNA expression level of different antioxidant enzymes was compared between cells that were cultured with NOX1 inhibitor (gray line) or without it (black line). Error bars represent S.D. ( b I) The percent of dead cells was evaluated by aqua blue staining. The division rate of untreated aASCs was compared with aASCs that were treated with different concentrations of NOX1 inhibitor ( b II), and their long-term propagation of cells was evaluated ( b III). ( b IV) The number of cells of untreated aASCs and aASCs treated with 2.5 μ M NOX1 inhibitor was compared at the indicated passages in two independent experiments. ( b V) Activities of caspase-3/7 in untreated aASCs and aASCs treated with 2.5 μ M were measured by a Caspase-Glo 3/7 Assay Kit. Error bars represent S.E.M. (Student's two-tailed t -test for equal ( b II) or unequal ( b IV) variance). ** P
Figure Legend Snippet: A specific NOX1 inhibitor reduces ROS accumulation and promotes long-term expansion of aASCs. Abdominal ASCs (Abd) were propagated under normoxic oxygen conditions (21% oxygen) in the presence of an NOX1-specific inhibitor ML171 (0, 0.5, 2.5 and 5 μ M). The following analyses were performed at the indicated passages (P). ( a I) ROS accumulation was evaluated by FACS (fluorescence-activated cell sorting) analysis using DCFDA staining, and the results of untreated aASCs compared with aASCs treated with 5 μ M inhibitor are shown. ( a II) Starting from P3, an additional population with a low DCFDA signal appeared (marked by an arrow). Dual staining of cells by DCFDA and aqua blue dye (an amine viability dye) was performed and analyzed by FACS. Results demonstrated that the majority of low DCFDA cells were aqua blue-positive dead cells. ( a III) RNA expression level of different antioxidant enzymes was compared between cells that were cultured with NOX1 inhibitor (gray line) or without it (black line). Error bars represent S.D. ( b I) The percent of dead cells was evaluated by aqua blue staining. The division rate of untreated aASCs was compared with aASCs that were treated with different concentrations of NOX1 inhibitor ( b II), and their long-term propagation of cells was evaluated ( b III). ( b IV) The number of cells of untreated aASCs and aASCs treated with 2.5 μ M NOX1 inhibitor was compared at the indicated passages in two independent experiments. ( b V) Activities of caspase-3/7 in untreated aASCs and aASCs treated with 2.5 μ M were measured by a Caspase-Glo 3/7 Assay Kit. Error bars represent S.E.M. (Student's two-tailed t -test for equal ( b II) or unequal ( b IV) variance). ** P

Techniques Used: FACS, Fluorescence, Staining, RNA Expression, Cell Culture, Caspase-Glo Assay, Two Tailed Test

scASCs display reduced NOX1 expression, reduced ROS accumulation and long-term expansion. aASCs (Abd) and scASCs (SC) were cultured under normoxic conditions (21% oxygen) and the following analyses were performed at the indicated passages (P). ( a ) ROS accumulation was detected by DCFDA staining and analyzed by FACS (fluorescence-activated cell sorting) ( a I) and by fluorescence microscopy ( a II). ( a III) The mRNA expression level of different antioxidant enzymes and various cytokines was evaluated by quantitative reverse transcription-PCR (qRT-PCR) at the indicated passages. Error bars represent S.D. ( b I) Cells at passages 3–5 were seeded at a density of 2 × 10 4 cells per 6 cm plate. The division rate was assessed by calculating the logarithmic growing phase. ( b II) Growth curves of ASCs from an abdominal fat source (dotted lines) and from subcutaneous fat (continuous line). Only scASCs cells were able to undergo long-term expansion. Experiments were performed on five independent preparations from each fat source. Error bars represent S.D. ** P
Figure Legend Snippet: scASCs display reduced NOX1 expression, reduced ROS accumulation and long-term expansion. aASCs (Abd) and scASCs (SC) were cultured under normoxic conditions (21% oxygen) and the following analyses were performed at the indicated passages (P). ( a ) ROS accumulation was detected by DCFDA staining and analyzed by FACS (fluorescence-activated cell sorting) ( a I) and by fluorescence microscopy ( a II). ( a III) The mRNA expression level of different antioxidant enzymes and various cytokines was evaluated by quantitative reverse transcription-PCR (qRT-PCR) at the indicated passages. Error bars represent S.D. ( b I) Cells at passages 3–5 were seeded at a density of 2 × 10 4 cells per 6 cm plate. The division rate was assessed by calculating the logarithmic growing phase. ( b II) Growth curves of ASCs from an abdominal fat source (dotted lines) and from subcutaneous fat (continuous line). Only scASCs cells were able to undergo long-term expansion. Experiments were performed on five independent preparations from each fat source. Error bars represent S.D. ** P

Techniques Used: Expressing, Cell Culture, Staining, FACS, Fluorescence, Microscopy, Polymerase Chain Reaction, Quantitative RT-PCR

Hypoxic conditions (3% oxygen) inhibit ROS accumulation by abdominal ASCs and allow their long-term propagation. Abdominal fat was isolated and ASCs were propagated under ‘normoxic' conditions (21% oxygen) or hypoxic conditions (3% oxygen). The following analyses were performed on aASCs that were grown under different oxygen conditions. ( a ) ROS accumulation was detected by DCFDA staining and analyzed by fluorescence microscopy or by FACS (fluorescence-activated cell sorting) ( a I and a II, respectively). ( b ) Growth curves of aASCs cultured at 3% oxygen (gray line) and 21% oxygen (black line). Only cells that were cultured at 3% oxygen were able to undergo long-term expansion. ( c and d ) RNA expression of various genes was compared by quantitative reverse transcription-PCR (qRT-PCR) analysis between cells that were grown under hypoxic conditions (gray line) to those grown under normoxic conditions (black line) at the indicated passages. ( e ) The expression of NOX1, NOX2 and NOX4 were evaluated in cells that were grown under hypoxic (gray) or normoxic (black) conditions by qRT-PCR. The expression is presented as RNA expression ( e I) or as the fold of expression between passage 5 and 2 ( e II). Error bars represent S.D. Results were repeated two times following the same trend
Figure Legend Snippet: Hypoxic conditions (3% oxygen) inhibit ROS accumulation by abdominal ASCs and allow their long-term propagation. Abdominal fat was isolated and ASCs were propagated under ‘normoxic' conditions (21% oxygen) or hypoxic conditions (3% oxygen). The following analyses were performed on aASCs that were grown under different oxygen conditions. ( a ) ROS accumulation was detected by DCFDA staining and analyzed by fluorescence microscopy or by FACS (fluorescence-activated cell sorting) ( a I and a II, respectively). ( b ) Growth curves of aASCs cultured at 3% oxygen (gray line) and 21% oxygen (black line). Only cells that were cultured at 3% oxygen were able to undergo long-term expansion. ( c and d ) RNA expression of various genes was compared by quantitative reverse transcription-PCR (qRT-PCR) analysis between cells that were grown under hypoxic conditions (gray line) to those grown under normoxic conditions (black line) at the indicated passages. ( e ) The expression of NOX1, NOX2 and NOX4 were evaluated in cells that were grown under hypoxic (gray) or normoxic (black) conditions by qRT-PCR. The expression is presented as RNA expression ( e I) or as the fold of expression between passage 5 and 2 ( e II). Error bars represent S.D. Results were repeated two times following the same trend

Techniques Used: Isolation, Staining, Fluorescence, Microscopy, FACS, Cell Culture, RNA Expression, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

10) Product Images from "Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition"

Article Title: Ca2+ binding to F‐ATP synthase β subunit triggers the mitochondrial permeability transition

Journal: EMBO Reports

doi: 10.15252/embr.201643354

T163S β subunit mutation protects against cell death caused by ionomycin and arachidonic acid in HeLa cells Adherent cells expressing WT or T163S (T > S) β subunit were incubated in HBSS containing 1 mM Ca 2+ after loading with either Fluo4‐FF or TMRM. Upper panels show images of cells stained with TMRM. In the lower panels, Ca 2+ (Fluo‐4FF) or mitochondrial membrane potential (TMRM) was monitored. Where indicated, 2 μM ionomycin (Iono) and 5 μM FCCP (F) were added. Fluorescence is expressed as % of the value reached after the addition of 5 μM digitonin (Fluo‐4FF) or of the value at the moment of ionomycin addition (TMRM). In the lower panels, one representative experiment of 3 ± s.d. is reported. Adherent cells expressing WT or T163S (T > S) β subunit were treated for 2 h with 2 μM ionomycin and lysates evaluated by Western blotting for β subunit (β) and caspase (Casp) 3 (the cleaved form is indicated by an arrow). Molecular size is indicated on the right. Detached WT and (T > S) β‐expressing cells were treated with 2 μM ionomycin for up to 40 min in the presence of 0.6% (v/v) FLUOS‐conjugated Annexin‐V and 2 μM propidium iodide (PI) and cell death was assessed by flow cytometry. Percentage of dead cells was assessed by FACS at 0, 20, and 40 min of ionomycin treatment. Data are mean of three independent experiment ± s.e. * P = 0.044, Student's t ‐test. A representative experiment of cells treated as in (C) and monitored by FACS is presented showing healthy (black), PI‐positive (red), Annexin‐V‐positive (green), and PI + Annexin‐V‐positive (blue) cells after 40 min of treatment. Dead cell percentage is shown on the right, mean of six independent experiments ± s.e. ** P = 0.0050, Student's t ‐test. Cell death was monitored by FACS analysis in detached cells treated with 200 μM arachidonic acid for 2 h in a medium containing Annexin‐V and PI. Data on the right are mean of six independent experiments ± s.e. ** P = 0.0038, Student's t ‐test.
Figure Legend Snippet: T163S β subunit mutation protects against cell death caused by ionomycin and arachidonic acid in HeLa cells Adherent cells expressing WT or T163S (T > S) β subunit were incubated in HBSS containing 1 mM Ca 2+ after loading with either Fluo4‐FF or TMRM. Upper panels show images of cells stained with TMRM. In the lower panels, Ca 2+ (Fluo‐4FF) or mitochondrial membrane potential (TMRM) was monitored. Where indicated, 2 μM ionomycin (Iono) and 5 μM FCCP (F) were added. Fluorescence is expressed as % of the value reached after the addition of 5 μM digitonin (Fluo‐4FF) or of the value at the moment of ionomycin addition (TMRM). In the lower panels, one representative experiment of 3 ± s.d. is reported. Adherent cells expressing WT or T163S (T > S) β subunit were treated for 2 h with 2 μM ionomycin and lysates evaluated by Western blotting for β subunit (β) and caspase (Casp) 3 (the cleaved form is indicated by an arrow). Molecular size is indicated on the right. Detached WT and (T > S) β‐expressing cells were treated with 2 μM ionomycin for up to 40 min in the presence of 0.6% (v/v) FLUOS‐conjugated Annexin‐V and 2 μM propidium iodide (PI) and cell death was assessed by flow cytometry. Percentage of dead cells was assessed by FACS at 0, 20, and 40 min of ionomycin treatment. Data are mean of three independent experiment ± s.e. * P = 0.044, Student's t ‐test. A representative experiment of cells treated as in (C) and monitored by FACS is presented showing healthy (black), PI‐positive (red), Annexin‐V‐positive (green), and PI + Annexin‐V‐positive (blue) cells after 40 min of treatment. Dead cell percentage is shown on the right, mean of six independent experiments ± s.e. ** P = 0.0050, Student's t ‐test. Cell death was monitored by FACS analysis in detached cells treated with 200 μM arachidonic acid for 2 h in a medium containing Annexin‐V and PI. Data on the right are mean of six independent experiments ± s.e. ** P = 0.0038, Student's t ‐test.

Techniques Used: Mutagenesis, Expressing, Incubation, Staining, Fluorescence, Western Blot, Flow Cytometry, Cytometry, FACS

11) Product Images from "Dendritic Cell Targeting Effectively Boosts T Cell Responses Elicited by an HIV Multiepitope DNA Vaccine"

Article Title: Dendritic Cell Targeting Effectively Boosts T Cell Responses Elicited by an HIV Multiepitope DNA Vaccine

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00101

The chimeric αDECHIVBr8 was successfully produced and retained its ability to bind to cells expressing the DEC205 receptors . (A) One microgram of each monoclonal antibody (mAb) was run on 12% SDS-PAGE under reducing conditions. An immunoblot was performed using peroxidase-labeled goat anti-mouse IgG Fc specific and peroxidase-labeled goat anti-mouse IgG kappa. Molecular weight (kilodaltons), αDEC (control), and αDECHIVBr8; (B) CHO cells expressing either DEC205 (left) or DCIR2 (right) receptors were incubated with 4, 2, or 1 µg/mL of αDEC (control) or αDECHIVBr8, following staining with anti-mouse IgG1 PE antibody. Fifty thousand events were acquired in FACS Canto II and analysis was performed using FlowJo software; (C) 5 million splenocytes from BALB/c mice were incubated with 4, 2, or 1 µg/mL of the chimeric αDECHIVBr8 or αDEC mAbs. Splenocytes were then incubated with a pool of fluorescent antibodies and gated as singlets and CD3 − CD19 − CD49b − . Dendritic cells were selected as CD11c + IAIE + and subsequently divided into CD8α + and CD8α − . Binding was detected on 3 × 10 6 cells using an anti-mouse IgG1-PE antibody. Analysis was performed using FlowJo software.
Figure Legend Snippet: The chimeric αDECHIVBr8 was successfully produced and retained its ability to bind to cells expressing the DEC205 receptors . (A) One microgram of each monoclonal antibody (mAb) was run on 12% SDS-PAGE under reducing conditions. An immunoblot was performed using peroxidase-labeled goat anti-mouse IgG Fc specific and peroxidase-labeled goat anti-mouse IgG kappa. Molecular weight (kilodaltons), αDEC (control), and αDECHIVBr8; (B) CHO cells expressing either DEC205 (left) or DCIR2 (right) receptors were incubated with 4, 2, or 1 µg/mL of αDEC (control) or αDECHIVBr8, following staining with anti-mouse IgG1 PE antibody. Fifty thousand events were acquired in FACS Canto II and analysis was performed using FlowJo software; (C) 5 million splenocytes from BALB/c mice were incubated with 4, 2, or 1 µg/mL of the chimeric αDECHIVBr8 or αDEC mAbs. Splenocytes were then incubated with a pool of fluorescent antibodies and gated as singlets and CD3 − CD19 − CD49b − . Dendritic cells were selected as CD11c + IAIE + and subsequently divided into CD8α + and CD8α − . Binding was detected on 3 × 10 6 cells using an anti-mouse IgG1-PE antibody. Analysis was performed using FlowJo software.

Techniques Used: Produced, Expressing, SDS Page, Labeling, Molecular Weight, Incubation, Staining, FACS, Software, Mouse Assay, Binding Assay

12) Product Images from "Long-term exposure to decabrominated diphenyl ether impairs CD8 T-cell function in adult mice"

Article Title: Long-term exposure to decabrominated diphenyl ether impairs CD8 T-cell function in adult mice

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2014.16

Continuous BDE-209 exposure impaired proliferation and induced apoptosis of CD8 T cells. ( a ) PBMCs from control mice and mice administered BDE-209 at a dose of 800 mg/kg bw were labeled with CFSE (5 µM) and stimulated with pre-bound anti-CD3e (2 µg/ml) plus soluble anti-CD28 (5 µg/ml) for 0, 48 or 72 h. The proliferation of CD8 T cells was visualized by FACS analysis of CFSE fluorescence in CD8 + cells (solid lines, control mice; dotted lines, BDE-209-exposed mice). ( b , c ) Mice were intragastrically administered BDE-209 at a dose of 8, 80 or 800 mg/kg bw at 2-day intervals, and splenic CD8 T cells were purified from the control mice and BDE-209-exposed mice using a FACS Aria II based on the surface expression of CD3 and CD8 (CD3 + CD8 + ) at month 3. The purified splenic-CD8 T cells were labeled with CFSE (5 µM) and then stimulated with anti-CD3 and anti-CD28 mAbs coupled to magnetic beads at a cell-bead ratio of 4∶1 for 72 h (dotted lines, unstimulated splenic CD8 + T cells). The proliferation ( b ) and the proportions of early (Annexin V + PI − ) and late apoptotic or necrotic (Annexin V + PI + ) cells ( c ) were evaluated by FACS analysis of the CD8 + cells. Each group included five mice; representative images are shown. BDE, brominated diphenyl ether; bw, body weight; PBMC, peripheral blood mononuclear cell.
Figure Legend Snippet: Continuous BDE-209 exposure impaired proliferation and induced apoptosis of CD8 T cells. ( a ) PBMCs from control mice and mice administered BDE-209 at a dose of 800 mg/kg bw were labeled with CFSE (5 µM) and stimulated with pre-bound anti-CD3e (2 µg/ml) plus soluble anti-CD28 (5 µg/ml) for 0, 48 or 72 h. The proliferation of CD8 T cells was visualized by FACS analysis of CFSE fluorescence in CD8 + cells (solid lines, control mice; dotted lines, BDE-209-exposed mice). ( b , c ) Mice were intragastrically administered BDE-209 at a dose of 8, 80 or 800 mg/kg bw at 2-day intervals, and splenic CD8 T cells were purified from the control mice and BDE-209-exposed mice using a FACS Aria II based on the surface expression of CD3 and CD8 (CD3 + CD8 + ) at month 3. The purified splenic-CD8 T cells were labeled with CFSE (5 µM) and then stimulated with anti-CD3 and anti-CD28 mAbs coupled to magnetic beads at a cell-bead ratio of 4∶1 for 72 h (dotted lines, unstimulated splenic CD8 + T cells). The proliferation ( b ) and the proportions of early (Annexin V + PI − ) and late apoptotic or necrotic (Annexin V + PI + ) cells ( c ) were evaluated by FACS analysis of the CD8 + cells. Each group included five mice; representative images are shown. BDE, brominated diphenyl ether; bw, body weight; PBMC, peripheral blood mononuclear cell.

Techniques Used: Mouse Assay, Labeling, FACS, Fluorescence, Purification, Expressing, Magnetic Beads

13) Product Images from "Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators"

Article Title: Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192717

DOX regulates inflammatory responses and tissue degrading enzyme activity in brain cells of PbA infected animals. C57BL/6 mice were left naïve or infected with PbA as described above and indicated groups received DOX (80 mg/kg/day) from dpi 4–6. Six days post PbA infection, brain tissue of naïve mice and PbA infected mice ± DOX was examined for inflammatory responses and MMP activity. (A) RNA was prepared from brain tissue and analyzed via PCR array. Volcano plot analysis of the comparison PbA versus PbA DOX: Shown is the logarithmic fold-change (x-axis) against the negative logarithmic p-value (y-axis). The dashed lines indicate where p ≤ 0.05 and FC ≥2. (B) Additionally, IFN-γ was analyzed by RT-qPCR in the brain tissue of all groups (C) Quantification of MMP-2-specific zymography. Brain tissue extracted on dpi 6 was subjected to zymography and quantified by scanning densitometry of the gelatinolytic bands of MMP-2. Representative experiments are shown with 6–7 mice/ group. Relative scanning units of MMP-2 are shown as fold change against expression of naïve animals displayed as median (D) Characterization of brain leukocytes on dpi 6 from naïve, PbA and PbA+DOX-treated animals. Flow cytometry gating scheme to identify CD8 + T cells is shown for representative animals from naïve and infected ± 80 mg/kg/day DOX mice. (E) Absolute numbers of brain infiltrating CD45 hi CD8a + cells as determined by FACS. (F) Brain tissue supernatant from dpi 6 was analyzed with an ELISA for granzyme B. (B, C, E, F) *p
Figure Legend Snippet: DOX regulates inflammatory responses and tissue degrading enzyme activity in brain cells of PbA infected animals. C57BL/6 mice were left naïve or infected with PbA as described above and indicated groups received DOX (80 mg/kg/day) from dpi 4–6. Six days post PbA infection, brain tissue of naïve mice and PbA infected mice ± DOX was examined for inflammatory responses and MMP activity. (A) RNA was prepared from brain tissue and analyzed via PCR array. Volcano plot analysis of the comparison PbA versus PbA DOX: Shown is the logarithmic fold-change (x-axis) against the negative logarithmic p-value (y-axis). The dashed lines indicate where p ≤ 0.05 and FC ≥2. (B) Additionally, IFN-γ was analyzed by RT-qPCR in the brain tissue of all groups (C) Quantification of MMP-2-specific zymography. Brain tissue extracted on dpi 6 was subjected to zymography and quantified by scanning densitometry of the gelatinolytic bands of MMP-2. Representative experiments are shown with 6–7 mice/ group. Relative scanning units of MMP-2 are shown as fold change against expression of naïve animals displayed as median (D) Characterization of brain leukocytes on dpi 6 from naïve, PbA and PbA+DOX-treated animals. Flow cytometry gating scheme to identify CD8 + T cells is shown for representative animals from naïve and infected ± 80 mg/kg/day DOX mice. (E) Absolute numbers of brain infiltrating CD45 hi CD8a + cells as determined by FACS. (F) Brain tissue supernatant from dpi 6 was analyzed with an ELISA for granzyme B. (B, C, E, F) *p

Techniques Used: Activity Assay, Infection, Mouse Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Zymography, Expressing, Flow Cytometry, Cytometry, FACS, Enzyme-linked Immunosorbent Assay

14) Product Images from "Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi"

Article Title: Identification of a Golgi-localized UDP-N-acetylglucosamine transporter in Trypanosoma cruzi

Journal: BMC Microbiology

doi: 10.1186/s12866-015-0601-7

Identification of a UDP-GlcNAc transporter (TcNST1) of T. cruzi by in vivo complementation assays. K. lactis mutant (Kl3) cells were transfected with TcNST1, the K. lactis UDP-GlcNAc transporter (Kl UGT, positive control) or empty vector (pE4, negative control). Wild-type K. lactis cells were included in the analysis for comparison. Cells were grown as described in the Methods section. After labeling with GS-II lectin (Alexa Fluor 488 conjugate), yeast cells were separated by flow cytometry in a FACS Canto II (Becton Dickinson). a Representative histograms showing lectin binding transfected cells. b Mean fluorescence intensities of a representative experiment in triplicate. Error bars represent the standard deviations. * p
Figure Legend Snippet: Identification of a UDP-GlcNAc transporter (TcNST1) of T. cruzi by in vivo complementation assays. K. lactis mutant (Kl3) cells were transfected with TcNST1, the K. lactis UDP-GlcNAc transporter (Kl UGT, positive control) or empty vector (pE4, negative control). Wild-type K. lactis cells were included in the analysis for comparison. Cells were grown as described in the Methods section. After labeling with GS-II lectin (Alexa Fluor 488 conjugate), yeast cells were separated by flow cytometry in a FACS Canto II (Becton Dickinson). a Representative histograms showing lectin binding transfected cells. b Mean fluorescence intensities of a representative experiment in triplicate. Error bars represent the standard deviations. * p

Techniques Used: In Vivo, Mutagenesis, Transfection, Positive Control, Plasmid Preparation, Negative Control, Labeling, Flow Cytometry, Cytometry, FACS, Binding Assay, Fluorescence

15) Product Images from "Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110"

Article Title: Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141669

Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM + CHO cells and different combinations of EpCAM + CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3 + T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM + CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC 50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3 . Error bars represent SEM values from two independent experiments
Figure Legend Snippet: Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM + CHO cells and different combinations of EpCAM + CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3 + T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM + CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC 50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3 . Error bars represent SEM values from two independent experiments

Techniques Used: Lysis, Expressing, Cell Culture, FACS, Software

16) Product Images from "Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation"

Article Title: Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0227932

Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto II. The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 –filtered HBS buffer without MPs (negative control); A2, B2 –MPs from erythrocytes; A3, B3 –MPs from platelets; A4, B4 –MPs from THP-1 cells; A5, B5 –MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) were counted in the SSC/FSC window (A1-A5) in the size gate
Figure Legend Snippet: Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto II. The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 –filtered HBS buffer without MPs (negative control); A2, B2 –MPs from erythrocytes; A3, B3 –MPs from platelets; A4, B4 –MPs from THP-1 cells; A5, B5 –MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) were counted in the SSC/FSC window (A1-A5) in the size gate

Techniques Used: Flow Cytometry, FACS, Fluorescence, Negative Control

17) Product Images from "Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators"

Article Title: Doxycycline inhibits experimental cerebral malaria by reducing inflammatory immune reactions and tissue-degrading mediators

Journal: PLoS ONE

doi: 10.1371/journal.pone.0192717

DOX regulates inflammatory responses and tissue degrading enzyme activity in brain cells of PbA infected animals. C57BL/6 mice were left naïve or infected with PbA as described above and indicated groups received DOX (80 mg/kg/day) from dpi 4–6. Six days post PbA infection, brain tissue of naïve mice and PbA infected mice ± DOX was examined for inflammatory responses and MMP activity. (A) RNA was prepared from brain tissue and analyzed via PCR array. Volcano plot analysis of the comparison PbA versus PbA DOX: Shown is the logarithmic fold-change (x-axis) against the negative logarithmic p-value (y-axis). The dashed lines indicate where p ≤ 0.05 and FC ≥2. (B) Additionally, IFN-γ was analyzed by RT-qPCR in the brain tissue of all groups (C) Quantification of MMP-2-specific zymography. Brain tissue extracted on dpi 6 was subjected to zymography and quantified by scanning densitometry of the gelatinolytic bands of MMP-2. Representative experiments are shown with 6–7 mice/ group. Relative scanning units of MMP-2 are shown as fold change against expression of naïve animals displayed as median (D) Characterization of brain leukocytes on dpi 6 from naïve, PbA and PbA+DOX-treated animals. Flow cytometry gating scheme to identify CD8 + T cells is shown for representative animals from naïve and infected ± 80 mg/kg/day DOX mice. (E) Absolute numbers of brain infiltrating CD45 hi CD8a + cells as determined by FACS. (F) Brain tissue supernatant from dpi 6 was analyzed with an ELISA for granzyme B. (B, C, E, F) *p
Figure Legend Snippet: DOX regulates inflammatory responses and tissue degrading enzyme activity in brain cells of PbA infected animals. C57BL/6 mice were left naïve or infected with PbA as described above and indicated groups received DOX (80 mg/kg/day) from dpi 4–6. Six days post PbA infection, brain tissue of naïve mice and PbA infected mice ± DOX was examined for inflammatory responses and MMP activity. (A) RNA was prepared from brain tissue and analyzed via PCR array. Volcano plot analysis of the comparison PbA versus PbA DOX: Shown is the logarithmic fold-change (x-axis) against the negative logarithmic p-value (y-axis). The dashed lines indicate where p ≤ 0.05 and FC ≥2. (B) Additionally, IFN-γ was analyzed by RT-qPCR in the brain tissue of all groups (C) Quantification of MMP-2-specific zymography. Brain tissue extracted on dpi 6 was subjected to zymography and quantified by scanning densitometry of the gelatinolytic bands of MMP-2. Representative experiments are shown with 6–7 mice/ group. Relative scanning units of MMP-2 are shown as fold change against expression of naïve animals displayed as median (D) Characterization of brain leukocytes on dpi 6 from naïve, PbA and PbA+DOX-treated animals. Flow cytometry gating scheme to identify CD8 + T cells is shown for representative animals from naïve and infected ± 80 mg/kg/day DOX mice. (E) Absolute numbers of brain infiltrating CD45 hi CD8a + cells as determined by FACS. (F) Brain tissue supernatant from dpi 6 was analyzed with an ELISA for granzyme B. (B, C, E, F) *p

Techniques Used: Activity Assay, Infection, Mouse Assay, Polymerase Chain Reaction, Quantitative RT-PCR, Zymography, Expressing, Flow Cytometry, Cytometry, FACS, Enzyme-linked Immunosorbent Assay

18) Product Images from "Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family"

Article Title: Insight into the mechanism of cytotoxicity of membrane-permeant psoralenic Kv1.3 channel inhibitors by chemical dissection of a novel member of the family

Journal: Redox Biology

doi: 10.1016/j.redox.2020.101705

PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p
Figure Legend Snippet: PAP-1-MHEG, a PAP-1 derivative with improved solubility, blocks cell proliferation and triggers apoptosis by inhibiting Kv1.3 in vitro and ex vivo in leukemia and melanoma cell lines. a) PAP-1 derivatives and their . chemical structures. b) Inhibitory effect of PAP-1 MHEG on three Kv channels expressed in CHO cells. Values are reported as means ± SEM (n = 4). c) Whole-cell Kv1.3 current traces recorded in a Jurkat lymphocyte. Currents were elicited by pulses to +70 mV (from a holding potential of −50 mV) before and after addition of PAP-1-MHEG. d) Growth curves of the cell proliferation assay. Human Jurkat leukemic T cells were chronically treated or not with the indicated concentrations of PAP-1 MHEG, and counted daily over a period of 4 days. Cells maintained in serum-free medium to block their proliferation were used as control. Values are reported as means of viable cells ± SEM (n = 3). e) Induction of apoptosis of B cells derived from CLL patients (n = 14) and from healthy donors (n = 5) by PAP-1 or PAP-1-MHEG, with or without CSH, after treatment for 24 h. 4 μM CSH was used as MDR inhibitor where indicated. Values are means ± SEM. f) Death of B-CLL cells co-cultured for 6 days with mesenchymal stromal cells (MSCs) to mimic the tumor microenvironment. Apoptosis was assessed by counting Annexin-V-positive cells by FACS. For all panels statistical significance (ANOVA or Student's t-test) was determined (* = p

Techniques Used: Solubility, In Vitro, Ex Vivo, Proliferation Assay, Blocking Assay, Derivative Assay, Cell Surface Hydrophobicity, Cell Culture, FACS

19) Product Images from "Lysophosphatidic acid enhances survival of human CD34+ cells in ischemic conditions"

Article Title: Lysophosphatidic acid enhances survival of human CD34+ cells in ischemic conditions

Journal: Scientific Reports

doi: 10.1038/srep16406

Proliferation and differentiation of LPA-treated CD34 + cells. ( A ) Number of non-treated and LPA-treated cells after 1 day in hypoxia and additional 3 (4 th day) or 6 days (7 th day) in normoxia. Cells were treated with 100 μM of LPA. ( B ) Gene expression of cyclin G ( CCNG2 ) as assessed by qRT-PCR analyses. Gene expression was normalized by the expression of GAPDH .( C ) CD34 + cell cycle, as evaluated by flow cytometry and PI staining, was performed in LPA- or non-treated cells. ( C.1 , C.2 ) show representative FACS results for each experimental group. ( D ) Cell differentiation as measured by flow cytometry using a cocktail of antibodies (please see Supplementary Table 2 ) to identify hematopoietic cell types. Cells were cultured in hypoxia for 1 day followed or not by 6 days in normoxia. In all graphs, cells were cultured with or without LPA (100 μM). In all studies were performed in CD34 + cells isolated from at least 3 different donors; 1–5 technical replicates. *Denotes statistical significance: * P
Figure Legend Snippet: Proliferation and differentiation of LPA-treated CD34 + cells. ( A ) Number of non-treated and LPA-treated cells after 1 day in hypoxia and additional 3 (4 th day) or 6 days (7 th day) in normoxia. Cells were treated with 100 μM of LPA. ( B ) Gene expression of cyclin G ( CCNG2 ) as assessed by qRT-PCR analyses. Gene expression was normalized by the expression of GAPDH .( C ) CD34 + cell cycle, as evaluated by flow cytometry and PI staining, was performed in LPA- or non-treated cells. ( C.1 , C.2 ) show representative FACS results for each experimental group. ( D ) Cell differentiation as measured by flow cytometry using a cocktail of antibodies (please see Supplementary Table 2 ) to identify hematopoietic cell types. Cells were cultured in hypoxia for 1 day followed or not by 6 days in normoxia. In all graphs, cells were cultured with or without LPA (100 μM). In all studies were performed in CD34 + cells isolated from at least 3 different donors; 1–5 technical replicates. *Denotes statistical significance: * P

Techniques Used: Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, FACS, Cell Differentiation, Cell Culture, Isolation

Survival mechanism in LPA-treated CD34 + cells. ( A ) Expression of LPA receptors. Cells were cultured in the different experimental conditions for 24 h. Gene expression was normalized by the expression of GAPDH .Studies were performed in CD34 + cells isolated from 3 different donors; 4 technical replicates. ( B ) Involvement of LPA receptors in cell survival. Cells were pre-treated with Rho kinase inhibitor (Y-2762), MEK1/2 inhibitor (PD98059), LPA 1 - and LPA 3 -specific inhibitor (Ki16425), a pan-G protein inhibitor (1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate (BrP)), or peroxisome proliferator-activator receptor γ (PPAR-γ) inhibitor (GW9662) for 1 h before hypoxia and cell medium containing LPA (100 μM) for 24 h. Cells without any pre-treatment and cultured in serum-free medium with or without LPA, in hypoxia for 24 h, were used as positive and negative controls, respectively. Studies were performed in CD34 + cells isolated from at least 3 different donors; 1–8 technical replicates. # and * means statistical difference relatively to cells without any pre-treatment and cultured in serum free medium without LPA, and with LPA, respectively. ( C ) Percentage of viable cells was measured by FACS. CD34 + cells in hypoxia and serum deprived conditions were treated with LPA, LPA with MEK1/2 inhibitor (PD98059; an upstream activator of MAPK/Erk), or LPA with Akt inhibitor (MK2206). Studies were performed in CD34 + cells isolated from 3 different donors. ( D ) Gene expression of VEGFA , BAX and CASP9 . NT and LPA mean non-treated and LPA-treated CD34 + cells in suspension. Gene expression was normalized by the expression of GAPDH .( E ) Caspase 9 activity in LPA-treated or non-treated CD34 + cells either in suspension (Cells) or encapsulated in a fibrin gel (Gels) at 24 h. Activity was assessed by Caspase Glo® 9 Assay kit. ( F ) Gene expression of BAX and CASP9 in fibrin gel encapsulated cells. Gene expression was normalized by the expression of GAPDH . In ( D – F ) studies were performed in CD34 + cells isolated from 3 different donors; 4 technical replicates. * ,# Denotes statistical significance: * ,# P
Figure Legend Snippet: Survival mechanism in LPA-treated CD34 + cells. ( A ) Expression of LPA receptors. Cells were cultured in the different experimental conditions for 24 h. Gene expression was normalized by the expression of GAPDH .Studies were performed in CD34 + cells isolated from 3 different donors; 4 technical replicates. ( B ) Involvement of LPA receptors in cell survival. Cells were pre-treated with Rho kinase inhibitor (Y-2762), MEK1/2 inhibitor (PD98059), LPA 1 - and LPA 3 -specific inhibitor (Ki16425), a pan-G protein inhibitor (1-bromo-3(S)-hydroxy-4-(palmitoyloxy)butyl]phosphonate (BrP)), or peroxisome proliferator-activator receptor γ (PPAR-γ) inhibitor (GW9662) for 1 h before hypoxia and cell medium containing LPA (100 μM) for 24 h. Cells without any pre-treatment and cultured in serum-free medium with or without LPA, in hypoxia for 24 h, were used as positive and negative controls, respectively. Studies were performed in CD34 + cells isolated from at least 3 different donors; 1–8 technical replicates. # and * means statistical difference relatively to cells without any pre-treatment and cultured in serum free medium without LPA, and with LPA, respectively. ( C ) Percentage of viable cells was measured by FACS. CD34 + cells in hypoxia and serum deprived conditions were treated with LPA, LPA with MEK1/2 inhibitor (PD98059; an upstream activator of MAPK/Erk), or LPA with Akt inhibitor (MK2206). Studies were performed in CD34 + cells isolated from 3 different donors. ( D ) Gene expression of VEGFA , BAX and CASP9 . NT and LPA mean non-treated and LPA-treated CD34 + cells in suspension. Gene expression was normalized by the expression of GAPDH .( E ) Caspase 9 activity in LPA-treated or non-treated CD34 + cells either in suspension (Cells) or encapsulated in a fibrin gel (Gels) at 24 h. Activity was assessed by Caspase Glo® 9 Assay kit. ( F ) Gene expression of BAX and CASP9 in fibrin gel encapsulated cells. Gene expression was normalized by the expression of GAPDH . In ( D – F ) studies were performed in CD34 + cells isolated from 3 different donors; 4 technical replicates. * ,# Denotes statistical significance: * ,# P

Techniques Used: Expressing, Cell Culture, Isolation, FACS, Activity Assay

20) Product Images from "Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110"

Article Title: Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141669

Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM + CHO cells and different combinations of EpCAM + CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3 + T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM + CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC 50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3 . Error bars represent SEM values from two independent experiments
Figure Legend Snippet: Combinatorial effect of evasion mechanisms on AMG 110-mediated redirected target cell lysis. Parental human EpCAM + CHO cells and different combinations of EpCAM + CHO cells expressing evasion proteins were co-cultured at an E:T ratio of 4:1 with CD3 + T cells and various AMG 110 concentrations for 72 h. For each curve an equal amount of control EpCAM + CHO cells was replaced by an additional stable CHO cell line until the entire target cell population comprised evasion protein-expressing cells and contained 1 mM adenosine. (A) Target cell lysis was determined by a FACS-based cytotoxicity assays. Sigmoidal dose-response curves were created with GraphPad Prism software. Error bars represent SEM values. (B) Relative change in EC 50 and (C) percentage of cell lysis after 72 h were calculated as described in Fig 3 . Error bars represent SEM values from two independent experiments

Techniques Used: Lysis, Expressing, Cell Culture, FACS, Software

21) Product Images from "Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia *"

Article Title: Proteomics and metabolomics identify molecular mechanisms of aging potentially predisposing for chronic lymphocytic leukemia *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.RA117.000425

Aging may predispose for CLL. ( A , B ) Principal component analysis of ( A ) cytoplasmic and ( B ) nuclear fractions of all four sample groups; a clear distinction between B cells from young donors, elderly donors, and CLL patients can be observed in the principal component analysis of the cytoplasmic fractions. ( C , D ) Selected proteins up-regulated ( C ) or down-regulated ( D ) in the cytoplasmic fractions of aged B cells from elderly donors and even more so in B cells from CLL patients are depicted. ( E-H ) Cytoplasmic proteins deregulated in elderly donors but hardly further altered in CLL. These proteins are related to ( E ) inflammatory response, ( F ) mitochondrial metabolism, ( G ) DNA damage response, and ( H ) mitochondrial stress and aging. ( I ) FACS-based ROS analyses of B cells, T cells, and monocytes obtained from young and elderly healthy donors as well as CLL patients. Statistical significance for displayed regulations was assessed by Student's t test with; *, p ≤ 0.05 and **, FDR≤0.05 applying multiparameter correction. Lines indicate mean label-free quantification values.
Figure Legend Snippet: Aging may predispose for CLL. ( A , B ) Principal component analysis of ( A ) cytoplasmic and ( B ) nuclear fractions of all four sample groups; a clear distinction between B cells from young donors, elderly donors, and CLL patients can be observed in the principal component analysis of the cytoplasmic fractions. ( C , D ) Selected proteins up-regulated ( C ) or down-regulated ( D ) in the cytoplasmic fractions of aged B cells from elderly donors and even more so in B cells from CLL patients are depicted. ( E-H ) Cytoplasmic proteins deregulated in elderly donors but hardly further altered in CLL. These proteins are related to ( E ) inflammatory response, ( F ) mitochondrial metabolism, ( G ) DNA damage response, and ( H ) mitochondrial stress and aging. ( I ) FACS-based ROS analyses of B cells, T cells, and monocytes obtained from young and elderly healthy donors as well as CLL patients. Statistical significance for displayed regulations was assessed by Student's t test with; *, p ≤ 0.05 and **, FDR≤0.05 applying multiparameter correction. Lines indicate mean label-free quantification values.

Techniques Used: FACS

22) Product Images from "Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10"

Article Title: Autoreactivity and Exceptional CDR Plasticity (but Not Unusual Polyspecificity) Hinder Elicitation of the Anti-HIV Antibody 4E10

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003639

Analysis of 4E10H knock-in mice. ( A ) The percentages of IgM + IgD − pre-B and IgM + IgD + immature B cells within the B220 + cell population in the bone marrow of B6 WT, B6-KL25H and B6-4E10H mice are shown. Marrow cells were isolated and stained for cell surface markers as described in the methods section. FACS plots were previously gated B220 + and on live cell size based on forward and side scatter. Numbers represent the percentages of B220 + bone marrow cells falling within each box gate. ( B ) The proliferation of B220 + splenocytes from B6 WT, B6-KL25H, and B6-4E10H mice in response to overnight culture in the presence of B cell stimuli are shown. Cells were loaded with cell proliferation dye before overnight incubation, and proliferation was assessed by FACS analysis after cell surface staining to identify B220 + B cells. Histograms were previously gated B220 + and on live cell size based on forward and side scatter. Numbers represent the percentage of B220 + cells falling within the bar gate on each plot, indicating dilution of the proliferation dye as a result of cell division.
Figure Legend Snippet: Analysis of 4E10H knock-in mice. ( A ) The percentages of IgM + IgD − pre-B and IgM + IgD + immature B cells within the B220 + cell population in the bone marrow of B6 WT, B6-KL25H and B6-4E10H mice are shown. Marrow cells were isolated and stained for cell surface markers as described in the methods section. FACS plots were previously gated B220 + and on live cell size based on forward and side scatter. Numbers represent the percentages of B220 + bone marrow cells falling within each box gate. ( B ) The proliferation of B220 + splenocytes from B6 WT, B6-KL25H, and B6-4E10H mice in response to overnight culture in the presence of B cell stimuli are shown. Cells were loaded with cell proliferation dye before overnight incubation, and proliferation was assessed by FACS analysis after cell surface staining to identify B220 + B cells. Histograms were previously gated B220 + and on live cell size based on forward and side scatter. Numbers represent the percentage of B220 + cells falling within the bar gate on each plot, indicating dilution of the proliferation dye as a result of cell division.

Techniques Used: Knock-In, Mouse Assay, Isolation, Staining, FACS, Incubation

23) Product Images from "Mitochondrial dysfunction reduces yeast replicative lifespan by elevating RAS-dependent ROS production by the ER-localized NADPH oxidase Yno1"

Article Title: Mitochondrial dysfunction reduces yeast replicative lifespan by elevating RAS-dependent ROS production by the ER-localized NADPH oxidase Yno1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198619

RAS signaling pathway and Yno1 contribute to the buildup of intracellular ROS in cells lacking mitochondrial respiration. (A, E, and H) Intracellular ROS levels in the indicated strains were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P
Figure Legend Snippet: RAS signaling pathway and Yno1 contribute to the buildup of intracellular ROS in cells lacking mitochondrial respiration. (A, E, and H) Intracellular ROS levels in the indicated strains were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P

Techniques Used: Fluorescence, FACS, Flow Cytometry, Cytometry, Standard Deviation

Increased ROS level decreases RLS in respiratory-deficient cells. (A) Intracellular ROS levels in wild-type (WT), rho 0 , cyc3 Δ, shy1 Δ, cox5a Δ, cyc1 Δ, and WT cells treated with 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM) were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. (B) Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P
Figure Legend Snippet: Increased ROS level decreases RLS in respiratory-deficient cells. (A) Intracellular ROS levels in wild-type (WT), rho 0 , cyc3 Δ, shy1 Δ, cox5a Δ, cyc1 Δ, and WT cells treated with 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM) were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. (B) Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P

Techniques Used: Fluorescence, FACS, Flow Cytometry, Cytometry, Standard Deviation

24) Product Images from "Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling"

Article Title: Human pluripotent stem cell derived midbrain PITX3eGFP/w neurons: a versatile tool for pharmacological screening and neurodegenerative modeling

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2015.00104

Transcriptional identity of PITX3 eGFP/w cells is midbrain dopaminergic. A second flow cytometry experiment was conducted to collect eGFP + and eGFP - cells for total RNA extraction and qPCR analysis on days 20, 40, 60, and 80. (A) Shows a typical FACS plot at day 20, and the strategy used to isolate eGFP + and eGFP - cells. This graph shows the percentage of live cells (from three independent experiments; ± SEM) sorted into the eGFP + gate at each time point. (B) qPCR analysis of the mRNA from at least 10 6 eGFP + cells showed upregulation of neuronal and midbrain dopaminergic genes (compared to hPSCs; one-way ANOVA with post hoc Dunnett’s test, p
Figure Legend Snippet: Transcriptional identity of PITX3 eGFP/w cells is midbrain dopaminergic. A second flow cytometry experiment was conducted to collect eGFP + and eGFP - cells for total RNA extraction and qPCR analysis on days 20, 40, 60, and 80. (A) Shows a typical FACS plot at day 20, and the strategy used to isolate eGFP + and eGFP - cells. This graph shows the percentage of live cells (from three independent experiments; ± SEM) sorted into the eGFP + gate at each time point. (B) qPCR analysis of the mRNA from at least 10 6 eGFP + cells showed upregulation of neuronal and midbrain dopaminergic genes (compared to hPSCs; one-way ANOVA with post hoc Dunnett’s test, p

Techniques Used: Flow Cytometry, RNA Extraction, Real-time Polymerase Chain Reaction, FACS

25) Product Images from "A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity"

Article Title: A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

Journal: Oncotarget

doi:

C4 CAR T cells secrete Th1 proinflammatory cytokine in response to tumor cell line and primary ovarian cancer cell surface associated αFR A. C4-27z CAR but not UNT T cells (10 5 cells/well) secret IFN-γ following overnight incubation with ovarian and breast cancer cell lines (10 5 cells/well) expressing different levels of surface αFR. Mean IFN-γ concentration ± SEM (pg/ml) from triplicate cultures is shown. B. Correlation between αFR expression (mean fluorescence intensity; MFI) on tumor cells and the production of IFN-γ by C4-27z CAR T cells cocultured with these tumor cells was plotted. C. Three primary ovarian cancer solid tumor samples (PT#1909, 1911, and 1913) and two ascites samples (PT#1801 and 1888) gated on CD45- EpCAM+ cells were stained with anti-αFR antibody (open histogram) or matched isotype controls (filled gray histogram) and analyzed by flow cytometry. A1847 and C30 cell lines served as αFR positive and negative expression controls, respectively. D. C4-27z CAR-modified T cells secrete IFN-γ during overnight culture with CD45-depleted αFR-expressing primary human ovarian cancer samples and αFR pos control cell line A1847, but not αFR neg C30 ovarian cancer cells. E. Antigen-specific T-cell induction of surface CD137 (4-1BB) expression was measured by FACS detection on C4-27z CAR T cells when stimulated overnight with αFR+ tumor cells (SKOV3, A1847 or T47D) but not αFR neg cells. UNT T cells did not upregulate CD137 expression. CD137 expression was preferentially increased on C4 CAR+ T-cell populations but not C4 CAR-T cells, after stimulation with αFR pos tumor cells. Representative dotplots from flow-cytometric analysis of CAR T cells stimulated with SKOV3 αFR pos tumor cells are shown and results were similar using all αFR pos tumor cells tested.
Figure Legend Snippet: C4 CAR T cells secrete Th1 proinflammatory cytokine in response to tumor cell line and primary ovarian cancer cell surface associated αFR A. C4-27z CAR but not UNT T cells (10 5 cells/well) secret IFN-γ following overnight incubation with ovarian and breast cancer cell lines (10 5 cells/well) expressing different levels of surface αFR. Mean IFN-γ concentration ± SEM (pg/ml) from triplicate cultures is shown. B. Correlation between αFR expression (mean fluorescence intensity; MFI) on tumor cells and the production of IFN-γ by C4-27z CAR T cells cocultured with these tumor cells was plotted. C. Three primary ovarian cancer solid tumor samples (PT#1909, 1911, and 1913) and two ascites samples (PT#1801 and 1888) gated on CD45- EpCAM+ cells were stained with anti-αFR antibody (open histogram) or matched isotype controls (filled gray histogram) and analyzed by flow cytometry. A1847 and C30 cell lines served as αFR positive and negative expression controls, respectively. D. C4-27z CAR-modified T cells secrete IFN-γ during overnight culture with CD45-depleted αFR-expressing primary human ovarian cancer samples and αFR pos control cell line A1847, but not αFR neg C30 ovarian cancer cells. E. Antigen-specific T-cell induction of surface CD137 (4-1BB) expression was measured by FACS detection on C4-27z CAR T cells when stimulated overnight with αFR+ tumor cells (SKOV3, A1847 or T47D) but not αFR neg cells. UNT T cells did not upregulate CD137 expression. CD137 expression was preferentially increased on C4 CAR+ T-cell populations but not C4 CAR-T cells, after stimulation with αFR pos tumor cells. Representative dotplots from flow-cytometric analysis of CAR T cells stimulated with SKOV3 αFR pos tumor cells are shown and results were similar using all αFR pos tumor cells tested.

Techniques Used: Incubation, Expressing, Concentration Assay, Fluorescence, Staining, Flow Cytometry, Cytometry, Modification, FACS

26) Product Images from "Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes"

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00525

Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p
Figure Legend Snippet: Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

Techniques Used: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).
Figure Legend Snippet: Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

Techniques Used: Activity Assay, Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p
Figure Legend Snippet: Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

Techniques Used: Transgenic Assay, Mouse Assay, Injection, Negative Control, Staining, FACS

27) Product Images from "MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her‐2/PI3K/AKT Signaling, MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her‐2/PI3K/AKT Signaling"

Article Title: MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her‐2/PI3K/AKT Signaling, MLH1 Deficiency Induces Cetuximab Resistance in Colon Cancer via Her‐2/PI3K/AKT Signaling

Journal: Advanced Science

doi: 10.1002/advs.202000112

MLH1 depletion enhances CRC resistance to CTX by inducing the Her‐2/PI3K/AKT signaling pathway in vivo. HCT116 cells overexpressing MLH1 were treated with an activator of the PI3K/AKT signaling pathway, while MLH1 negative control HCT116 cells were transfected with shRNA‐Her‐2. MLH1‐knockdown Caco‐2 cells were treated with an inhibitor of the PI3K/AKT signaling pathway, while MLH1 negative control Caco‐2 cells were transfected with LV‐Her‐2. All CRC cells received CTX treatment for 48 h at the respective IC50 concentration unless otherwise indicated. A) CCK‐8 analysis of HCT116 cell proliferation with the above‐mentioned treatment. CRC cells were treated with the indicated treatments. B) Cells were treated with increasing concentrations of cetuximab (5, 10, 15, and 20 µg mL −1 ) for 48 h, and cell viability was determined by the CCK‐8 assay. C) FACS analysis of cell apoptosis in HCT116 and Caco‐2 cells with the above‐mentioned treatment. D) Caspase‐3 activation after CTX treatment, as indicated by green fluorescent staining. E) Expression of proapoptotic proteins (Bax and active caspase‐3) and an antiapoptotic protein (Bcl‐2) in CTX‐treated CRC cells. Data are presented as the mean ± SD from three independent experiments. * p
Figure Legend Snippet: MLH1 depletion enhances CRC resistance to CTX by inducing the Her‐2/PI3K/AKT signaling pathway in vivo. HCT116 cells overexpressing MLH1 were treated with an activator of the PI3K/AKT signaling pathway, while MLH1 negative control HCT116 cells were transfected with shRNA‐Her‐2. MLH1‐knockdown Caco‐2 cells were treated with an inhibitor of the PI3K/AKT signaling pathway, while MLH1 negative control Caco‐2 cells were transfected with LV‐Her‐2. All CRC cells received CTX treatment for 48 h at the respective IC50 concentration unless otherwise indicated. A) CCK‐8 analysis of HCT116 cell proliferation with the above‐mentioned treatment. CRC cells were treated with the indicated treatments. B) Cells were treated with increasing concentrations of cetuximab (5, 10, 15, and 20 µg mL −1 ) for 48 h, and cell viability was determined by the CCK‐8 assay. C) FACS analysis of cell apoptosis in HCT116 and Caco‐2 cells with the above‐mentioned treatment. D) Caspase‐3 activation after CTX treatment, as indicated by green fluorescent staining. E) Expression of proapoptotic proteins (Bax and active caspase‐3) and an antiapoptotic protein (Bcl‐2) in CTX‐treated CRC cells. Data are presented as the mean ± SD from three independent experiments. * p

Techniques Used: In Vivo, Negative Control, Transfection, shRNA, Concentration Assay, CCK-8 Assay, FACS, Activation Assay, Staining, Expressing

28) Product Images from "An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence"

Article Title: An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence

Journal: Scientific Reports

doi: 10.1038/srep43670

Resistance gene expression after polymyxin B selection at the single-cell level. ( A ) Representative scheme of the P pbgPE - gfp [AAV] transcriptional fusion between the pbgPE promoter and a destabilized GFP-encoding gene. A GFP-positive bacterium expressing resistance genes is shown. ( B ) TT01/P pbgPE - gfp [AAV]) was cultured in LB and samples taken over a time course were fixed with formaldehyde and analyzed in a FACS Canto II cytometer. Results are presented as dot plots of side scatter (FSC) against GFP fluorescence intensity. Each dot represents one bacterium. ( C ) TT01/P pbgPE - gfp [AAV] was first cultured in LB. Samples were collected at an OD 540 of 0.3, before and after the addition of polymyxin B to the culture medium. The representation used here is as in panel B. Only live cells were considered, after treatment with eFluor 660. One representative experiment from more than three independent experiments is shown.
Figure Legend Snippet: Resistance gene expression after polymyxin B selection at the single-cell level. ( A ) Representative scheme of the P pbgPE - gfp [AAV] transcriptional fusion between the pbgPE promoter and a destabilized GFP-encoding gene. A GFP-positive bacterium expressing resistance genes is shown. ( B ) TT01/P pbgPE - gfp [AAV]) was cultured in LB and samples taken over a time course were fixed with formaldehyde and analyzed in a FACS Canto II cytometer. Results are presented as dot plots of side scatter (FSC) against GFP fluorescence intensity. Each dot represents one bacterium. ( C ) TT01/P pbgPE - gfp [AAV] was first cultured in LB. Samples were collected at an OD 540 of 0.3, before and after the addition of polymyxin B to the culture medium. The representation used here is as in panel B. Only live cells were considered, after treatment with eFluor 660. One representative experiment from more than three independent experiments is shown.

Techniques Used: Expressing, Selection, Cell Culture, FACS, Cytometry, Fluorescence

29) Product Images from "Mitochondrial dysfunction reduces yeast replicative lifespan by elevating RAS-dependent ROS production by the ER-localized NADPH oxidase Yno1"

Article Title: Mitochondrial dysfunction reduces yeast replicative lifespan by elevating RAS-dependent ROS production by the ER-localized NADPH oxidase Yno1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198619

RAS signaling pathway and Yno1 contribute to the buildup of intracellular ROS in cells lacking mitochondrial respiration. (A, E, and H) Intracellular ROS levels in the indicated strains were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P
Figure Legend Snippet: RAS signaling pathway and Yno1 contribute to the buildup of intracellular ROS in cells lacking mitochondrial respiration. (A, E, and H) Intracellular ROS levels in the indicated strains were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P

Techniques Used: Fluorescence, FACS, Flow Cytometry, Cytometry, Standard Deviation

Increased ROS level decreases RLS in respiratory-deficient cells. (A) Intracellular ROS levels in wild-type (WT), rho 0 , cyc3 Δ, shy1 Δ, cox5a Δ, cyc1 Δ, and WT cells treated with 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM) were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. (B) Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P
Figure Legend Snippet: Increased ROS level decreases RLS in respiratory-deficient cells. (A) Intracellular ROS levels in wild-type (WT), rho 0 , cyc3 Δ, shy1 Δ, cox5a Δ, cyc1 Δ, and WT cells treated with 3 μg/ml antimycin A (AA) or 10 μg/ml oligomycin (OM) were detected with H 2 DCFDA. Fluorescence was analyzed using a BD FACS Canto II flow cytometer. (B) Cells with high ROS were calculated as a percentage of cells with higher fluorescence intensity than the maximum fluorescence intensity of control sample without the ROS indicator. Values represent the average of three independent experiments, and error bars indicate the standard deviation. All asterisks indicate P

Techniques Used: Fluorescence, FACS, Flow Cytometry, Cytometry, Standard Deviation

30) Product Images from "Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer"

Article Title: Tryptophan 2,3-dioxygenase (TDO)-reactive T cells differ in their functional characteristics in health and cancer

Journal: Oncoimmunology

doi: 10.4161/21624011.2014.968480

CD4 + T-cell responses against TDO. ( A ) PBMC from a MM patient (MM020) were stimulated once with either an irrelevant HIV peptide (top) or the long TDO 303-322 peptide (bottom) before being analyzed by intracellular IFNγ staining. FACS plots were gated on living CD8 + T cells (left) or CD4 + T cells (right). ( B ) PBMC from a MM patient (MM020) peptide were stimulated four times with DC pulsed with TDO 303-322 . The resulting T-cell culture was stimulated once with either an irrelevant long TDO peptide (TDO 118-137 (left) or the long TDO 303-322 peptide (right) before being analyzed by intracellular TNFα and IFNγ staining. ( C ) PBMC from a MM patient (MM020) peptide were stimulated with TDO 303-322 (left). The IFNγ + CD4 + T cells were sorted by FACS and expanded by REP. The resulting T-cell culture was stimulated once with either an irrelevant long TDO peptide (TDO 118-137 (left) or the long TDO 303-322 peptide (right) before being analyzed by intracellular TNFα and IFNγ staining. The T-cell culture was highly TDO 303-322 specific. ( D ) ELISPOT analysis of reactivity toward TDO 303-322 in two highly specific T-cell lines from two MM patients (MM014 and MM020). 10,000 T cells were analyzed with or without TDO 303-322 . The bars in the panel depict mean value of triplicate experiments, background subtracted (left). The well images are depicted for both T-cell cultures (right).
Figure Legend Snippet: CD4 + T-cell responses against TDO. ( A ) PBMC from a MM patient (MM020) were stimulated once with either an irrelevant HIV peptide (top) or the long TDO 303-322 peptide (bottom) before being analyzed by intracellular IFNγ staining. FACS plots were gated on living CD8 + T cells (left) or CD4 + T cells (right). ( B ) PBMC from a MM patient (MM020) peptide were stimulated four times with DC pulsed with TDO 303-322 . The resulting T-cell culture was stimulated once with either an irrelevant long TDO peptide (TDO 118-137 (left) or the long TDO 303-322 peptide (right) before being analyzed by intracellular TNFα and IFNγ staining. ( C ) PBMC from a MM patient (MM020) peptide were stimulated with TDO 303-322 (left). The IFNγ + CD4 + T cells were sorted by FACS and expanded by REP. The resulting T-cell culture was stimulated once with either an irrelevant long TDO peptide (TDO 118-137 (left) or the long TDO 303-322 peptide (right) before being analyzed by intracellular TNFα and IFNγ staining. The T-cell culture was highly TDO 303-322 specific. ( D ) ELISPOT analysis of reactivity toward TDO 303-322 in two highly specific T-cell lines from two MM patients (MM014 and MM020). 10,000 T cells were analyzed with or without TDO 303-322 . The bars in the panel depict mean value of triplicate experiments, background subtracted (left). The well images are depicted for both T-cell cultures (right).

Techniques Used: Staining, FACS, Cell Culture, Enzyme-linked Immunospot

31) Product Images from "Targeting T cell Bioenergetics by Modulating P-glycoprotein Selectively Depletes Alloreactive T cells to Prevent GVHD"

Article Title: Targeting T cell Bioenergetics by Modulating P-glycoprotein Selectively Depletes Alloreactive T cells to Prevent GVHD

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

doi: 10.4049/jimmunol.1402445

Bioenergetic profile of superantigen stimulated T cells resembles the Warburg Effect PBMCs were stimulated with SEB for 72 hours. (A) Activated (CD3 + CD25 + ) and resting (CD3 + CD25 − ) T cells were then identified by FACS analysis, as shown for one representative experiment. Activated and resting T cells were then isolated, and bioenergetics were measured by real-time extracellular flux analysis. The (B) oxygen consumption rate (OCR) and (C) spare respiratory capacity (SRC) were measured in a basal state, and after the addition of oligomycin (to block ATP synthesis), FCCP (to uncouple ATP synthesis from the electron transport chain), and rotenone (to block complex I of the electron transport chain), as shown for one representative experiment. (D) The mean fluorescent intensity (MFI) of MitoTracker Green, (E) the basal OCR, (F) the basal extracellular acidification rate (ECAR), and (G) the OCR/ECAR ratio are shown for both activated and resting cells. Mean ± SE are plotted from 5 independent experiments. * p
Figure Legend Snippet: Bioenergetic profile of superantigen stimulated T cells resembles the Warburg Effect PBMCs were stimulated with SEB for 72 hours. (A) Activated (CD3 + CD25 + ) and resting (CD3 + CD25 − ) T cells were then identified by FACS analysis, as shown for one representative experiment. Activated and resting T cells were then isolated, and bioenergetics were measured by real-time extracellular flux analysis. The (B) oxygen consumption rate (OCR) and (C) spare respiratory capacity (SRC) were measured in a basal state, and after the addition of oligomycin (to block ATP synthesis), FCCP (to uncouple ATP synthesis from the electron transport chain), and rotenone (to block complex I of the electron transport chain), as shown for one representative experiment. (D) The mean fluorescent intensity (MFI) of MitoTracker Green, (E) the basal OCR, (F) the basal extracellular acidification rate (ECAR), and (G) the OCR/ECAR ratio are shown for both activated and resting cells. Mean ± SE are plotted from 5 independent experiments. * p

Techniques Used: FACS, Isolation, Blocking Assay

Photosensitizers are preferentially retained in stimulated T cells Stimulated T cells by were identified by FACS analysis for CD3 + and CD25 + coexpression. Resting T cells were identified as CD3 + without expression of CD25. A) A representative histogram of photosensitizer fluorescence is shown. B) Bar graphs represent the ratio of the mean fluorescent intensity (MFI) of CD25 + /CD25 − T cells for the strong and weak P-gp stimulating photosensitizers. Mean ± SE are plotted from 3 independent experiments. ** p
Figure Legend Snippet: Photosensitizers are preferentially retained in stimulated T cells Stimulated T cells by were identified by FACS analysis for CD3 + and CD25 + coexpression. Resting T cells were identified as CD3 + without expression of CD25. A) A representative histogram of photosensitizer fluorescence is shown. B) Bar graphs represent the ratio of the mean fluorescent intensity (MFI) of CD25 + /CD25 − T cells for the strong and weak P-gp stimulating photosensitizers. Mean ± SE are plotted from 3 independent experiments. ** p

Techniques Used: FACS, Expressing, Fluorescence

32) Product Images from "Cul4 E3 ubiquitin ligase regulates ovarian cancer drug resistance by targeting the antiapoptotic protein BIRC3"

Article Title: Cul4 E3 ubiquitin ligase regulates ovarian cancer drug resistance by targeting the antiapoptotic protein BIRC3

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1200-y

CRL4 knockdown increases the apoptosis of A2780CP cells in response to cisplatin. a Flow cytometry analysis showing apoptosis of A2780CP cells transduced with virus containing scrambled shRNA (NT) or shRNA against DDB1 or Cul4A. Quantification data were averaged from three independent experiments. Apoptosis rate was the sum of the two quadrats on the right in each FACS figure. Data represent mean ± SD. Significance of differences was calculated using Student’s t test (* P
Figure Legend Snippet: CRL4 knockdown increases the apoptosis of A2780CP cells in response to cisplatin. a Flow cytometry analysis showing apoptosis of A2780CP cells transduced with virus containing scrambled shRNA (NT) or shRNA against DDB1 or Cul4A. Quantification data were averaged from three independent experiments. Apoptosis rate was the sum of the two quadrats on the right in each FACS figure. Data represent mean ± SD. Significance of differences was calculated using Student’s t test (* P

Techniques Used: Flow Cytometry, Cytometry, Transduction, shRNA, FACS

33) Product Images from "The Transcription Factor T-Bet Is Required for Optimal Type I Follicular Helper T Cell Maintenance During Acute Viral Infection"

Article Title: The Transcription Factor T-Bet Is Required for Optimal Type I Follicular Helper T Cell Maintenance During Acute Viral Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00606

T-bet promotes TFH cell maintenance. (A,B) WT or Tbx21 −/− SMARTA cells (CD45.1 + ) were transferred into naïve WT mice (CD45.2 + ), which would be infected with LCMV. Splenocytes were isolated and analyzed for TFH response at day2, 5 and 8 post infection. Flow cytometry of TFH cells derived from transferred SMARTA cells (A) , and the summary of the percentages and numbers of those cells (B) . (C,D) WT and iT-bet −/− mice were treated with Tamoxifen for 3 days before or after LCMV infection. Spleens were harvested at day 9 post infection. (C) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen before infection (day−3 to−1). (D) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen after infection (day 5–7). (E) Setup of CD4 + T cell transfer experiment. CD4 + T cells were purified form spleens of iT-bet −/− mice (CD45.2 + ) without Tamoxifen treatment and adoptively transferred into infection-matched recipient mice (CD45.1 + ). At day 8–10 post infection, recipient mice were treated with Tamoxifen or vehicle. Spleens were harvested at day 14 post infection. (F) Representative flow cytometry of TFH cells (left) and summary of TFH cell number (right) in Tamoxifen treated mice (iTbx21 −/− ) and vehicle treated mice (WT) was showed here. (G) Setup of TFH co-transfer experiments. WT SMARTA cells (CD45.1 + CD45.2 + ) and Tbx21 −/− SMARTA cells (CD45.1 − CD45.2 + ) were transferred into naïve B6 mice (CD45.1 + CD45.2 − ) separately before infecting recipients with LCMV. At day 6 post infection, WT SMARTA TFH cells and Tbx21 −/− SMARTA TFH cells were FACS sorted and mixed (about 1:1), then co-transferred into infection-matched B6 mice (CD45.1 + CD45.2 − ). Spleens were harvested and analyzed for transferred TFH cells at day 9 post infection. (H) Flow cytometry of sorted SMARTA TFH cells before (day 6 p.i.) and after (day 9 p.i.) co-transfer (left). The percentage of Tbx21 −/− SMARTA TFH cells in total donor TFH cells and the number of WT and Tbx21 −/− SMARTA TFH cells at day 9 post infection were summarized (right). Numbers adjacent to outlined areas in (A,C,D,F,H) indicate percent of each cell subset in parent subset. ns, not significant; * P
Figure Legend Snippet: T-bet promotes TFH cell maintenance. (A,B) WT or Tbx21 −/− SMARTA cells (CD45.1 + ) were transferred into naïve WT mice (CD45.2 + ), which would be infected with LCMV. Splenocytes were isolated and analyzed for TFH response at day2, 5 and 8 post infection. Flow cytometry of TFH cells derived from transferred SMARTA cells (A) , and the summary of the percentages and numbers of those cells (B) . (C,D) WT and iT-bet −/− mice were treated with Tamoxifen for 3 days before or after LCMV infection. Spleens were harvested at day 9 post infection. (C) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen before infection (day−3 to−1). (D) Flow cytometry of TFH cells (left) with its number (right) in mice treat with Tamoxifen after infection (day 5–7). (E) Setup of CD4 + T cell transfer experiment. CD4 + T cells were purified form spleens of iT-bet −/− mice (CD45.2 + ) without Tamoxifen treatment and adoptively transferred into infection-matched recipient mice (CD45.1 + ). At day 8–10 post infection, recipient mice were treated with Tamoxifen or vehicle. Spleens were harvested at day 14 post infection. (F) Representative flow cytometry of TFH cells (left) and summary of TFH cell number (right) in Tamoxifen treated mice (iTbx21 −/− ) and vehicle treated mice (WT) was showed here. (G) Setup of TFH co-transfer experiments. WT SMARTA cells (CD45.1 + CD45.2 + ) and Tbx21 −/− SMARTA cells (CD45.1 − CD45.2 + ) were transferred into naïve B6 mice (CD45.1 + CD45.2 − ) separately before infecting recipients with LCMV. At day 6 post infection, WT SMARTA TFH cells and Tbx21 −/− SMARTA TFH cells were FACS sorted and mixed (about 1:1), then co-transferred into infection-matched B6 mice (CD45.1 + CD45.2 − ). Spleens were harvested and analyzed for transferred TFH cells at day 9 post infection. (H) Flow cytometry of sorted SMARTA TFH cells before (day 6 p.i.) and after (day 9 p.i.) co-transfer (left). The percentage of Tbx21 −/− SMARTA TFH cells in total donor TFH cells and the number of WT and Tbx21 −/− SMARTA TFH cells at day 9 post infection were summarized (right). Numbers adjacent to outlined areas in (A,C,D,F,H) indicate percent of each cell subset in parent subset. ns, not significant; * P

Techniques Used: Mouse Assay, Infection, Isolation, Flow Cytometry, Cytometry, Derivative Assay, Purification, FACS

34) Product Images from "Exosomal micro RNAs as tumor markers in epithelial ovarian cancer"

Article Title: Exosomal micro RNAs as tumor markers in epithelial ovarian cancer

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12371

MiR‐200b affects cell proliferation and apoptosis. OVCAR 3 cells were transiently transfected with a negative control, mimic, or inhibitor of miR‐200b. After 24, 48, and 72 h, cells were treated with MTT . Cell proliferation after overexpression and inhibition of miR‐200b was measured at an absorbance of 540 nm. The standard deviations from triplicate experiments are indicated in the line chart (A). OVCAR 3 cells were additionally treated with the topoisomerase I inhibitor camptothecin and analyzed by a bright‐field microscopy (left) and on a FACS Canto II device (right) for apoptosis. In order to observe morphological features of apoptosis, cells were analyzed using 10× magnification. Cells were labeled with Annexin‐V‐ FITC and propidium iodide for FACS analyses. Cell fragments only positive for propidium iodide can be found in the upper left corner (Q1). Late apoptotic as well as necrotic cells can be found in the upper right corner (Q2), since they are positive for Annexin and propidium iodide. Living cells are negative for Annexin and propidium iodide and therefore can be found in the lower left corner (Q3). Early apoptotic cells are only positive for Annexin and located in the lower right corner (Q4). The size for each population (%) is given in the corresponding area (B). A bar chart summarizes the data on the apoptotic effect by miR‐200b as derived from the FACS Canto II device. Three independent experiments were performed. Error bars are presented as means ± SD . ANOVA Tukey's HSD tests were used to determine the significance ( P
Figure Legend Snippet: MiR‐200b affects cell proliferation and apoptosis. OVCAR 3 cells were transiently transfected with a negative control, mimic, or inhibitor of miR‐200b. After 24, 48, and 72 h, cells were treated with MTT . Cell proliferation after overexpression and inhibition of miR‐200b was measured at an absorbance of 540 nm. The standard deviations from triplicate experiments are indicated in the line chart (A). OVCAR 3 cells were additionally treated with the topoisomerase I inhibitor camptothecin and analyzed by a bright‐field microscopy (left) and on a FACS Canto II device (right) for apoptosis. In order to observe morphological features of apoptosis, cells were analyzed using 10× magnification. Cells were labeled with Annexin‐V‐ FITC and propidium iodide for FACS analyses. Cell fragments only positive for propidium iodide can be found in the upper left corner (Q1). Late apoptotic as well as necrotic cells can be found in the upper right corner (Q2), since they are positive for Annexin and propidium iodide. Living cells are negative for Annexin and propidium iodide and therefore can be found in the lower left corner (Q3). Early apoptotic cells are only positive for Annexin and located in the lower right corner (Q4). The size for each population (%) is given in the corresponding area (B). A bar chart summarizes the data on the apoptotic effect by miR‐200b as derived from the FACS Canto II device. Three independent experiments were performed. Error bars are presented as means ± SD . ANOVA Tukey's HSD tests were used to determine the significance ( P

Techniques Used: Transfection, Negative Control, MTT Assay, Over Expression, Inhibition, Microscopy, FACS, Labeling, Derivative Assay

35) Product Images from "Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells"

Article Title: Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S108694

rsv-NLC reduce DC activation in response to TNF-α. Notes: Differentiated DCs were stimulated as indicated, with TNF-α, in presence or absence of rsv-NLC for 72 h, before surface staining for CD1a and CD83 (top left), or CD11c and CD86 (bottom), or HLA-DR (top right). Samples were analyzed using a FACS Calibur flow cytometer. ( A ) Representative plots of a phenotypic profile. ( B ) Graphs depict isotype control-subtracted MFI of CD83 (top left), HLA-DR (top right), or CD86 (bottom), over seven independent experiments. Each dot represents a different donor, and the line represents the mean value. Data were parametric and statistical significance was calculated using repeated measures analysis of variance, followed by Bonferroni’s multiple comparison test (* P
Figure Legend Snippet: rsv-NLC reduce DC activation in response to TNF-α. Notes: Differentiated DCs were stimulated as indicated, with TNF-α, in presence or absence of rsv-NLC for 72 h, before surface staining for CD1a and CD83 (top left), or CD11c and CD86 (bottom), or HLA-DR (top right). Samples were analyzed using a FACS Calibur flow cytometer. ( A ) Representative plots of a phenotypic profile. ( B ) Graphs depict isotype control-subtracted MFI of CD83 (top left), HLA-DR (top right), or CD86 (bottom), over seven independent experiments. Each dot represents a different donor, and the line represents the mean value. Data were parametric and statistical significance was calculated using repeated measures analysis of variance, followed by Bonferroni’s multiple comparison test (* P

Techniques Used: Activation Assay, Staining, FACS, Flow Cytometry, Cytometry

No cytotoxicity was observed upon NLC internalization. Notes: ( A ) Representative images of DC morphology, upon NLC internalization, as analyzed by ImageStream X . FITC (green)-loaded NLC were internalized by DC, which were then labeled for CD1a (yellow) and nuclei (DRAQ5, red). The overlay of the three channels is presented and cell complexity, in terms of intracellular vesicles, can be observed in the darkfield channel (purple). ( B ) Differentiated DCs were incubated with the indicated volumes of NLC for 24 hours, before being stained for Annexin V and PI and analyzed using a FACS Calibur flow cytometer. Abbreviations: DC, dendritic cells; DRAQ5, deep red anthraquinone 5; FITC, fluorescein isothiocyanate; PI, propidium iodide; NLC, nanostructured lipid carriers.
Figure Legend Snippet: No cytotoxicity was observed upon NLC internalization. Notes: ( A ) Representative images of DC morphology, upon NLC internalization, as analyzed by ImageStream X . FITC (green)-loaded NLC were internalized by DC, which were then labeled for CD1a (yellow) and nuclei (DRAQ5, red). The overlay of the three channels is presented and cell complexity, in terms of intracellular vesicles, can be observed in the darkfield channel (purple). ( B ) Differentiated DCs were incubated with the indicated volumes of NLC for 24 hours, before being stained for Annexin V and PI and analyzed using a FACS Calibur flow cytometer. Abbreviations: DC, dendritic cells; DRAQ5, deep red anthraquinone 5; FITC, fluorescein isothiocyanate; PI, propidium iodide; NLC, nanostructured lipid carriers.

Techniques Used: Labeling, Incubation, Staining, FACS, Flow Cytometry, Cytometry

36) Product Images from "Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis"

Article Title: Activation of GPR55 increases neural stem cell proliferation and promotes early adult hippocampal neurogenesis

Journal: British Journal of Pharmacology

doi: 10.1111/bph.14387

Effects of GPR55 agonists on hNSC differentiation. hNSCs were cultured under differentiating conditions (maintenance medium without bFGF and EGF) and treated with either vehicle (0.01% DMSO), the selective GPR55 agonist ML184 (1 μM), selective antagonist ML193 (5 μM) or a combination of ML184 and ML193 for 10 days. Representative images of vehicle‐treated (A), ML184 (B), ML193 (C), ML184 + ML193 (D) and undifferentiated (E) cells stained for βIII‐tubulin (red) and DAPI (blue). Scale bars are 100 μm. (F, H) Representative histograms of flow cytometric analysis of βIII‐tubulin and S100β respectively. For flow cytometric analysis, cells were incubated with antibodies against human βIII‐tubulin (APC) and S100β (Alexa‐fluor 488). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analysed with FlowJo software. Treatment with ML184 increased the expression of βIII‐tubulin after 10 days, while ML193 attenuated these effects. ML193 treatment decreased the formation of neurons as compared to differentiated vehicle control. Quantitative analysis of βIII‐tubulin+ cells (G) and S100β + cells (I) as compared to differentiated control from flow cytometric analysis of five experiments. mRNA was derived from whole cells collected from each experiment and analysed via qPCR for neuronal markers (βIII‐tubulin, J; Map2, K) and astrocytic markers (S100β, L; GFAP, M). Data are presented as fold change of vehicle‐treated cells under differentiating conditions for 10 days. Individual data points represent separate, individual experiments. * P
Figure Legend Snippet: Effects of GPR55 agonists on hNSC differentiation. hNSCs were cultured under differentiating conditions (maintenance medium without bFGF and EGF) and treated with either vehicle (0.01% DMSO), the selective GPR55 agonist ML184 (1 μM), selective antagonist ML193 (5 μM) or a combination of ML184 and ML193 for 10 days. Representative images of vehicle‐treated (A), ML184 (B), ML193 (C), ML184 + ML193 (D) and undifferentiated (E) cells stained for βIII‐tubulin (red) and DAPI (blue). Scale bars are 100 μm. (F, H) Representative histograms of flow cytometric analysis of βIII‐tubulin and S100β respectively. For flow cytometric analysis, cells were incubated with antibodies against human βIII‐tubulin (APC) and S100β (Alexa‐fluor 488). Cytometric acquisition was performed using a BD FACS Canto II flow cytometer and analysed with FlowJo software. Treatment with ML184 increased the expression of βIII‐tubulin after 10 days, while ML193 attenuated these effects. ML193 treatment decreased the formation of neurons as compared to differentiated vehicle control. Quantitative analysis of βIII‐tubulin+ cells (G) and S100β + cells (I) as compared to differentiated control from flow cytometric analysis of five experiments. mRNA was derived from whole cells collected from each experiment and analysed via qPCR for neuronal markers (βIII‐tubulin, J; Map2, K) and astrocytic markers (S100β, L; GFAP, M). Data are presented as fold change of vehicle‐treated cells under differentiating conditions for 10 days. Individual data points represent separate, individual experiments. * P

Techniques Used: Cell Culture, Staining, Flow Cytometry, Incubation, FACS, Cytometry, Software, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction

37) Product Images from "Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells"

Article Title: Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

Journal: Scientific Reports

doi: 10.1038/srep37289

Performance of the Lent-On-Plus LVs on sorted hMSCs. ( a ) Plots showing eGFP expression before (top plots) and after (bottom plots) purification of the eGFP+ populations of CEETnl2 and CEETnl2Is2-transduced hMSCs using fluorescence-activated cell sorting (FACS) Aria II Flow Cytometer. A MOI = 0.4 was used to keep the percentage of transduced hMSCs below 15%. ( b ) After sorting, the hMSCs were kept in the absence of Dox for 8 days and then induced (+Dox) or not (−Dox) for additional 5 days and analyzed for eGFP expression. The Percentages (%) of eGFP+ cells in the different population are shown inside each plot. The MFI of the whole living cells (WLC) (used to measure fold induction) are indicated at the bottom of each plot. Fold induction and leaking of the CEETnl2- and CEETnl2Is2-transduced sorted populations are indicated in the table (bottom).
Figure Legend Snippet: Performance of the Lent-On-Plus LVs on sorted hMSCs. ( a ) Plots showing eGFP expression before (top plots) and after (bottom plots) purification of the eGFP+ populations of CEETnl2 and CEETnl2Is2-transduced hMSCs using fluorescence-activated cell sorting (FACS) Aria II Flow Cytometer. A MOI = 0.4 was used to keep the percentage of transduced hMSCs below 15%. ( b ) After sorting, the hMSCs were kept in the absence of Dox for 8 days and then induced (+Dox) or not (−Dox) for additional 5 days and analyzed for eGFP expression. The Percentages (%) of eGFP+ cells in the different population are shown inside each plot. The MFI of the whole living cells (WLC) (used to measure fold induction) are indicated at the bottom of each plot. Fold induction and leaking of the CEETnl2- and CEETnl2Is2-transduced sorted populations are indicated in the table (bottom).

Techniques Used: Expressing, Purification, Fluorescence, FACS, Flow Cytometry, Cytometry

Performance of the Lent-On-Plus LVs on sorted hESCs. ( a ) Plots showing eGFP expression before (Pre-sorting) and after (Post-sorting) purification of the eGFP+ populations of CEETnl2 and CEETnl2Is2-transduced hESCs using fluorescence-activated cell sorting (FACS) Aria II Flow Cytometer. A MOI = 30 was used to achieve over 35% eGFP+ cells and good expression levels. ( b ) Sorted hESCs were kept in the absence of Dox for 8 days and then induced (+Dox) or not (−Dox) for additional 5 days and analyzed for eGFP expression. The Percentages (%) of eGFP+ cells are shown inside each plot. The MFI of the whole living cells (WLC) (used to measure fold induction) are indicated at the bottom of each plot. Fold induction and leaking of the CEETnl2- and CEETnl2Is2-transduced sorted populations are indicated in the table (bottom).
Figure Legend Snippet: Performance of the Lent-On-Plus LVs on sorted hESCs. ( a ) Plots showing eGFP expression before (Pre-sorting) and after (Post-sorting) purification of the eGFP+ populations of CEETnl2 and CEETnl2Is2-transduced hESCs using fluorescence-activated cell sorting (FACS) Aria II Flow Cytometer. A MOI = 30 was used to achieve over 35% eGFP+ cells and good expression levels. ( b ) Sorted hESCs were kept in the absence of Dox for 8 days and then induced (+Dox) or not (−Dox) for additional 5 days and analyzed for eGFP expression. The Percentages (%) of eGFP+ cells are shown inside each plot. The MFI of the whole living cells (WLC) (used to measure fold induction) are indicated at the bottom of each plot. Fold induction and leaking of the CEETnl2- and CEETnl2Is2-transduced sorted populations are indicated in the table (bottom).

Techniques Used: Expressing, Purification, Fluorescence, FACS, Flow Cytometry, Cytometry

38) Product Images from "CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas"

Article Title: CXCR4 upregulation is an indicator of sensitivity to B-cell receptor/PI3K blockade and a potential resistance mechanism in B-cell receptor-dependent diffuse large B-cell lymphomas

Journal: Haematologica

doi: 10.3324/haematol.2019.216218

PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P
Figure Legend Snippet: PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG-mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS-sorted for GFP expression, treated with 1 μM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 μM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT-PCR relative to PPIA. The P -values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t -test. *** P

Techniques Used: Expressing, Transduction, Plasmid Preparation, FACS, Flow Cytometry, Quantitative RT-PCR

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Article Title: Alcohol Induced Mitochondrial Oxidative Stress and Alveolar Macrophage Dysfunction
Article Snippet: Measurement of Intracellular ROS Generation After EtOH exposure, the cellular ROS sensitive probe CM-H2 DCFDA (Invitrogen; Carlsbad, CA) and the mitochondrial superoxide probe mitoSOX (Invitrogen; Carlsbad, CA) were added to the medium (10 μ M, 30 min, 37°C). .. Cells were then harvested, washed, and resuspended in phosphate buffered saline (PBS) for FACS analysis by BD Canto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). ..

Article Title: Epigenetic inhibitors eliminate senescent melanoma BRAFV600E cells that survive long-term BRAF inhibition
Article Snippet: At day 10, cells were fixed with 4% paraformaldehyde at room temperature for 15 min and stained with 0.05% cristal violet solution at room temperature for 20 min. .. Analysis of apoptosis and cell cycle Cell cycle and apoptosis were analysed using BD FACS Canto II or BD FACScalibur flow cytometers (both BD Biosciences). ..

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes
Article Snippet: Cytokine transport was blocked by incubating with Golgi-plug (BD Biosciences) for the last 4 h. Cells were stained with anti-mouse CD8-FITC (BD Biosciences), anti-mouse CD4-PE (BD Biosciences), and Live-dead-Aqua (Invitrogen), fixated with fixation/permeabilization buffer (BD Biosciences), and washed with permeabilization wash buffer (BD Biosciences). .. Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences). .. Acquired data were analyzed with FlowJo version 10 for Mac OSX (TreeStar Inc.).

Flow Cytometry:

Article Title: Alcohol Induced Mitochondrial Oxidative Stress and Alveolar Macrophage Dysfunction
Article Snippet: Measurement of Intracellular ROS Generation After EtOH exposure, the cellular ROS sensitive probe CM-H2 DCFDA (Invitrogen; Carlsbad, CA) and the mitochondrial superoxide probe mitoSOX (Invitrogen; Carlsbad, CA) were added to the medium (10 μ M, 30 min, 37°C). .. Cells were then harvested, washed, and resuspended in phosphate buffered saline (PBS) for FACS analysis by BD Canto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). ..

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes
Article Snippet: Cytokine transport was blocked by incubating with Golgi-plug (BD Biosciences) for the last 4 h. Cells were stained with anti-mouse CD8-FITC (BD Biosciences), anti-mouse CD4-PE (BD Biosciences), and Live-dead-Aqua (Invitrogen), fixated with fixation/permeabilization buffer (BD Biosciences), and washed with permeabilization wash buffer (BD Biosciences). .. Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences). .. Acquired data were analyzed with FlowJo version 10 for Mac OSX (TreeStar Inc.).

Article Title: A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining
Article Snippet: Flow cytometry analysis To prove that this method for counting oocysts without staining is independent of the model of flow cytometer, we ran the same samples on 3 flow cytometers to compare the efficiency of the gating strategy. .. Samples were analyzed using a BD LSRFortessa, a BD LSRFortessa X-20, and a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). ..

Cytometry:

Article Title: Alcohol Induced Mitochondrial Oxidative Stress and Alveolar Macrophage Dysfunction
Article Snippet: Measurement of Intracellular ROS Generation After EtOH exposure, the cellular ROS sensitive probe CM-H2 DCFDA (Invitrogen; Carlsbad, CA) and the mitochondrial superoxide probe mitoSOX (Invitrogen; Carlsbad, CA) were added to the medium (10 μ M, 30 min, 37°C). .. Cells were then harvested, washed, and resuspended in phosphate buffered saline (PBS) for FACS analysis by BD Canto II Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ). ..

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes
Article Snippet: Cytokine transport was blocked by incubating with Golgi-plug (BD Biosciences) for the last 4 h. Cells were stained with anti-mouse CD8-FITC (BD Biosciences), anti-mouse CD4-PE (BD Biosciences), and Live-dead-Aqua (Invitrogen), fixated with fixation/permeabilization buffer (BD Biosciences), and washed with permeabilization wash buffer (BD Biosciences). .. Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences). .. Acquired data were analyzed with FlowJo version 10 for Mac OSX (TreeStar Inc.).

Article Title: A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining
Article Snippet: Flow cytometry analysis To prove that this method for counting oocysts without staining is independent of the model of flow cytometer, we ran the same samples on 3 flow cytometers to compare the efficiency of the gating strategy. .. Samples were analyzed using a BD LSRFortessa, a BD LSRFortessa X-20, and a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). ..

Staining:

Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes
Article Snippet: Cytokine transport was blocked by incubating with Golgi-plug (BD Biosciences) for the last 4 h. Cells were stained with anti-mouse CD8-FITC (BD Biosciences), anti-mouse CD4-PE (BD Biosciences), and Live-dead-Aqua (Invitrogen), fixated with fixation/permeabilization buffer (BD Biosciences), and washed with permeabilization wash buffer (BD Biosciences). .. Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences). .. Acquired data were analyzed with FlowJo version 10 for Mac OSX (TreeStar Inc.).

Article Title: SARS-CoV-2-specific T cell memory is long-lasting in the majority of convalsecent COVID-19 individuals
Article Snippet: For surface staining, cells were incubated with relevant fluorochrome-labeled antibodies for 30 min at 4°C in the dark. .. For intracellular cytokine staining, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (Invitrogen, USA) and stained with FITC-anti-IFN-γ, PE-anti-IL-2 and APC-anti-TNF-α (BD Biosciences, USA). .. Approximately 100,000 PBMCs were acquired for each sample using a BD FACS Canto II flow cytometer.

Article Title: Unveiling the Immunomodulatory Characteristics of Haemonchus contortus Ephrin Domain Containing Protein in the Parasite–Host Interactions
Article Snippet: Afterwards, cells were centrifuged at 500 rcf for 5 min at 4 °C, added 500 μL of Fixation and permeabilization solution (BD Pharmingen, San Jose, CA, USA) for 20 min at 4 °C. .. Consequently, cells were stained with anti-goat intracellular cytokine monoclonal antibodies, IL-9 (PE-cy5; BD Pharmingen) and IL-10 (PE; BD Pharmingen) in addition to evaluate the expression of Th9 cells. .. Finally, flow cytometry was performed (setting the gate on CD2 + CD4 + T cell) by using intracellular antibodies (IL-9 and IL-10) to count the Th9 cells, acquiring the gate at 1000, on FACS Canto II flow cytometer (Becton Dickinson).

Software:

Article Title: A protocol to count Cryptosporidium oocysts by flow cytometry without antibody staining
Article Snippet: Flow cytometry analysis To prove that this method for counting oocysts without staining is independent of the model of flow cytometer, we ran the same samples on 3 flow cytometers to compare the efficiency of the gating strategy. .. Samples were analyzed using a BD LSRFortessa, a BD LSRFortessa X-20, and a BD FACSCanto II flow cytometer using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). ..

Ex Vivo:

Article Title: Cytotoxicity of CD56-positive lymphocytes against autologous B-cell precursor acute lymphoblastic leukemia cells
Article Snippet: Cells were analyzed on a FACS Canto II flow cytometer (BD Biosciences). .. For analysis of CD107a and IFNγ, ex vivo expanded NK cells (1 x 106 ) from ALL patient samples were stimulated with nothing, or with US7 cells (2x105 ) in the presence or absence of 10 μg/ml human control IgG Ab or αBAFF-R mAb as indicated for 1 hr, with inclusion of CD107a-PE antibodies (BD Bioscience, San Jose, CA). .. Non-expanded PBMCs were stimulated with PMA (2.5 μg/ml) and ionomycin (1.0 μg/ml) as a positive control.

Expressing:

Article Title: Unveiling the Immunomodulatory Characteristics of Haemonchus contortus Ephrin Domain Containing Protein in the Parasite–Host Interactions
Article Snippet: Afterwards, cells were centrifuged at 500 rcf for 5 min at 4 °C, added 500 μL of Fixation and permeabilization solution (BD Pharmingen, San Jose, CA, USA) for 20 min at 4 °C. .. Consequently, cells were stained with anti-goat intracellular cytokine monoclonal antibodies, IL-9 (PE-cy5; BD Pharmingen) and IL-10 (PE; BD Pharmingen) in addition to evaluate the expression of Th9 cells. .. Finally, flow cytometry was performed (setting the gate on CD2 + CD4 + T cell) by using intracellular antibodies (IL-9 and IL-10) to count the Th9 cells, acquiring the gate at 1000, on FACS Canto II flow cytometer (Becton Dickinson).

High Throughput Screening Assay:

Article Title: Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris
Article Snippet: Estimation of µ (specific growth rate) from OD750 or cell concentration data was carried out using an exponential least squares fit (GraphPad Prism; model y = y0 e µt where y0 is the initial value of y, t is time and µ is the rate constant i.e. specific growth rate) with manual inspection of semilog plots to verify fitting accuracy. .. Flow cytometry Cytometric analysis was carried out using a BD FACS Canto II Flow Cytometer (BD, San Jose, USA) fitted with a high throughput system (HTS) to analyse multiwell plate samples. .. Cell sorting was carried out using a BD FACS Aria cytometer.

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    Becton Dickinson facs canto ii flow cytometer
    Flow <t>cytometry</t> pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting <t>(FACS)</t> of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.
    Facs Canto Ii Flow Cytometer, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

    Journal: PLoS ONE

    Article Title: Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

    doi: 10.1371/journal.pone.0097269

    Figure Lengend Snippet: Flow cytometry pulse width measurements correlate well with cell diameter. Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

    Article Snippet: Flow cytometry Cytometric analysis was carried out using a BD FACS Canto II Flow Cytometer (BD, San Jose, USA) fitted with a high throughput system (HTS) to analyse multiwell plate samples.

    Techniques: Flow Cytometry, Cytometry, Fluorescence, FACS, Marker, Software, Standard Deviation

    Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Induction of GIL-specific T cell responses by different concentrations of GIL and whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with indicated amounts of GIL peptide and WIV. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (B) Splenocytes of immunized mice were re-stimulated with GIL peptide. The number of CD8 + IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of three mice per group, each dot is a single replicate and data are from a single experiment. * p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Comparison of CD8 + T cell responses induced by 1 nmol GIL peptide adjuvanted with either 50 µg CpG or 50 µg whole-inactivated influenza virus (WIV). HLA-A2.1 transgenic mice were injected twice, 3 weeks apart with PBS (negative control), WIV or GIL peptide with indicated adjuvant. Shown are the responses 2 weeks after the final immunization. (A) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. Data are shown as mean ± SD of three mice per group, each circle is a single replicate and data are from a single experiment representative of two individual experiments. ** p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Injection, Negative Control, Staining, FACS

    Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Influence of co-localization on the immunogenicity of GIL (100 nmol) and whole-inactivated influenza virus (WIV, 25 µg). (A) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide and WIV either combined in one single flank (mixed) or separate in opposite flanks (separate). (B) Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (A,B) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. ** p

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

    Journal: Frontiers in Immunology

    Article Title: Whole-Inactivated Influenza Virus Is a Potent Adjuvant for Influenza Peptides Containing CD8+ T Cell Epitopes

    doi: 10.3389/fimmu.2018.00525

    Figure Lengend Snippet: Effect of membrane fusion activity on whole-inactivated influenza virus (WIV) adjuvant activity. (A) WIV was fusion-inactivated by brief exposure to a buffer with pH 4.5 at 37°C. Hemolysis was performed by mixing either WIV (“active” WIV) or fusion-inactivated WIV (“inactive” WIV) with human blood erythrocytes at various pHs. Fusion-mediated hemoglobin release was subsequently determined by spectrophotometric analysis. Data are shown as mean ± SD of three individual experiments. (B) HLA-A2.1 transgenic mice were vaccinated twice, 3 weeks apart with GIL peptide (100 µg) and either fusion-active or fusion-inactive WIV (25 µg). Splenocytes were re-stimulated with GIL peptide and stained for intracellular cytokine IFN-γ. The percentages for IFN-γ + CD8 + T cells acquired by FACS are plotted. (C) Splenocytes were re-stimulated with GIL peptide. The number of IFN-γ-secreting cells was subsequently determined with ELISpot. (B,C) Data are shown as mean ± SD of six mice per group, each dot is a single replicate and data are from a single experiment. n.s. = not significant (one-way ANOVA).

    Article Snippet: Finally, cells were stained intracellularly with anti-mouse IFN-γ-APC (BD Biosciences), and IFN-γ+ CD8+ T cells were quantified on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Activity Assay, Transgenic Assay, Mouse Assay, Staining, FACS, Enzyme-linked Immunospot

    Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 – filtered HBS buffer without MPs (negative control); A2, B2 – MPs from erythrocytes; A3, B3 – MPs from platelets; A4, B4 – MPs from THP-1 cells; A5, B5 – MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) (gate P3 in B1-B5) were counted in the SSC/FSC window (A1-A5) in the size gate

    Journal: bioRxiv

    Article Title: Platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation

    doi: 10.1101/2020.01.06.895722

    Figure Lengend Snippet: Analysis and counting of MPs of different cellular origins by flow cytometry on a FACS Canto The noise threshold was set up in the FITC fluorescence channel (200 a.u) (B1-B5). A1, B1 – filtered HBS buffer without MPs (negative control); A2, B2 – MPs from erythrocytes; A3, B3 – MPs from platelets; A4, B4 – MPs from THP-1 cells; A5, B5 – MPs from ECs. Annexin V-FITC was added to all probes. All events above the FITC fluorescence threshold (200 a.u) (gate P3 in B1-B5) were counted in the SSC/FSC window (A1-A5) in the size gate

    Article Snippet: MPs were analysed and counted in a FACS Canto II flow cytometer (Becton Dickinson, BD Bioscience, San Jose, CA, USA).

    Techniques: Flow Cytometry, FACS, Fluorescence, Negative Control

    Comparison of the immunomodulatory capacity of CD105 - and CD105 + mASCs. (A) The levels of PGE 2 and TGF-β1 were measured in supernatants from ASC tot , CD105 - and CD105 + mASCs using specific ELISAs (see materials and methods). (B) ASC tot , CD105 - and CD105 + mASCs were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR. (C) Increasing numbers of mitomycin C-treated ASC tot , CD105 - and CD105 + mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 µg/ml) for 3 days. The cells were harvested and acquired on a FACS Canto II flow cytometer and the proliferation of CD4 + splenocytes was quantified using the FlowJo software. (D) BM-MΦs were cultured with or without mASC for 48 hours and then restimulated with LPS (1 µg/ml) for 24 hours. The levels of IL-10 (left graph) and IL-12 (right graph) in the supernatants of the co-cultures were measured using specific ELISAs. Data is shown as mean (SEM) of at least 3 independent experiments. *=p

    Journal: PLoS ONE

    Article Title: CD105 (Endoglin)-Negative Murine Mesenchymal Stromal Cells Define a New Multipotent Subpopulation with Distinct Differentiation and Immunomodulatory Capacities

    doi: 10.1371/journal.pone.0076979

    Figure Lengend Snippet: Comparison of the immunomodulatory capacity of CD105 - and CD105 + mASCs. (A) The levels of PGE 2 and TGF-β1 were measured in supernatants from ASC tot , CD105 - and CD105 + mASCs using specific ELISAs (see materials and methods). (B) ASC tot , CD105 - and CD105 + mASCs were stimulated with TNF-α (10 ng/ml) and IFN-γ (10 ng/ml) for 12 and 24 hours. Total RNA was purified for each time point and the expression of iNOS and IL-6 was analyzed using qPCR. (C) Increasing numbers of mitomycin C-treated ASC tot , CD105 - and CD105 + mASCs were cultured together with CFSE-labeled splenocytes (200,000 cells/well) and stimulated with anti-CD3 (1 µg/ml) for 3 days. The cells were harvested and acquired on a FACS Canto II flow cytometer and the proliferation of CD4 + splenocytes was quantified using the FlowJo software. (D) BM-MΦs were cultured with or without mASC for 48 hours and then restimulated with LPS (1 µg/ml) for 24 hours. The levels of IL-10 (left graph) and IL-12 (right graph) in the supernatants of the co-cultures were measured using specific ELISAs. Data is shown as mean (SEM) of at least 3 independent experiments. *=p

    Article Snippet: Cells were harvested on day 3, stained with anti-CD4-APC Abs and analyzed on a FACS Canto II flow cytometer (BD Biosciences).

    Techniques: Purification, Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Labeling, FACS, Flow Cytometry, Cytometry, Software