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BioLegend flow cytometry
Flow Cytometry, supplied by BioLegend, used in various techniques. Bioz Stars score: 95/100, based on 2323 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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flow cytometry - by Bioz Stars, 2020-09
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Article Title: Differential Immune Activation in Fetal Macrophage Populations
Article Snippet: Flow cytometry and cell sorting Isolated single cell suspensions were first incubated with FC block and Zombie NIR or Aqua live/dead stain (Biolegend) for 15 min in PBS.

Article Title: A T Cell Suppressive Circuitry Mediated by CD39 and Regulated by ShcC/Rai Is Induced in Astrocytes by Encephalitogenic T Cells
Article Snippet: Flow Cytometry and Proliferation Assays Flow cytometric analysis of astrocytes, MOG-T cells and splenocytes was performed using AlexaFluor488-, PE-, PerCP-conjugated anti-mouse antibodies to: GFAP (clone GA5; eBioscence), CD39 (clone 24DMS1; eBioscence), CD73 (clone TY11.8; Biolegend), CTLA-4 (clone UC10-4B9; Biolegend), IL-17A (clone TC11-18H10; Becton Dickinson), IFN-γ (clone XMG1.2; Becton Dickinson), GM-CSF (clone MPI-22E9; Biolegend), TNFα (Clone MP6-XT-22; Biolegend), and isotype control antibodies.

Article Title: PIK3IP1/TrIP restricts activation of T cells through inhibition of PI3K/Akt
Article Snippet: 1 d after transfection, cells were evaluated by flow cytometry for GFP expression and TrIP expression (by anti-Flag staining) using anti-DYKDDDDK APC clone L5 (BioLegend; 637308).

Article Title: Impact of human sepsis on CCCTC-binding factor associated monocyte transcriptional response of Major Histocompatibility Complex II components
Article Snippet: Magnetic cell sorting (autoMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using CD14 and CD16 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) was performed to deplete CD16++ monocytes and to isolate CD14++ CD16— monocytes, followed by flow cytometry after incubation with FITC anti-human CD14 Antibody (BioLegend, San Diego, USA) to ensure purity of the isolated cells.

Article Title: Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection
Article Snippet: CFSE-stained CD4 T cells were injected intravenously into recipient mice, spleens harvested 3 days later, and flow cytometry performed using allophycocyanin-conjugated anti-CD4 plus PE-conjugated anti-CD90.1/Thy1.1 (clone OX-7, BioLegend, San Diego, CA, USA) to identify live (7-AAD− , BD Biosciences) TCR75 T cells.

Article Title: Microparticle alpha-2-macroglobulin enhances pro-resolving responses and promotes survival in sepsis
Article Snippet: Flow cytometry and Western Blotting To determine microparticle levels in healthy volunteer and sepsis patient plasma samples microparticles were double stained using an anti-CD66b PE-conjugated antibody (1:25; clone G10F5; BioLegend, Cambridge UK) and Alexa488 conjugated anti-A2MG (5 μg/ml; Clone 3D1; Thermo Scientific, Northumberland, UK).

Article Title: Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies
Article Snippet: Immunophenotyping Gene-modified T cells were analyzed for transgene expression 10–14 days post-transduction by flow cytometry using CD3-PerCP.Cy5 (Biolegend Cat:317336) and CD34-PE or APC (Abnova Cat:MAB6483, R & D Systems Cat:FAB7227A).

Article Title: Coinfection with Leishmania major and Staphylococcus aureus enhances the pathologic responses to both microbes through a pathway involving IL-17A
Article Snippet: Within 10 minutes prior to analysis by flow cytometry, 10 μL of Propidium Iodide Staining Solution (Biolegend) was added to each sample.

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  • 88
    BioLegend cell surface flow cytometry
    Phenotypic analysis of natural killer (NK) and Hodgkin lymphoma (HL) cells. a NK-92, haNK, and primary NK cells were tested for CD16 expression by flow <t>cytometry.</t> Gating for CD56-positive cells and CD16 expression on the each of the NK cells was compared to a murine isotype control. Additionally, HL cell lines (KM-H2, L-428, and L-540), NK cells (NK-92, haNK, and primary NK cells), and the K562 control cell line were tested for cell surface CD123. b Results of the HL cell lines as (%) expression. c Relative variation of the median fluorescence intensity (RMFI) to a murine isotype control in all cells
    Cell Surface Flow Cytometry, supplied by BioLegend, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell surface flow cytometry/product/BioLegend
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    cell surface flow cytometry - by Bioz Stars, 2020-09
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    93
    BioLegend flow cytometry analysis antibodies against cd4
    VD restrained Th17 cells differentiation through miR-124 mediated inhibition of IL-6 signaling. <t>CD4</t> + CD62L + cells from C57BL/6J were cultured in the Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 with or without VD. In some experiments, naïve CD4 + T cells were transduced with 10 nM miR-124 inhibitor for 24 h using Lipofectamine® 3000 before polarized into Th17 cells. (A) qRT-PCR showed that VD upregulates miR-124 expression in Th17 cells (72 h). (B–D) naïve CD4 + T cells were transduced with miR-124 inhibitor (i) or inhibitor control (iNC) before Th17-polarization using immobilized anti-CD3 or APCs system and flow <t>cytometry</t> was used to confirm IL-17 expression at protein level. Data are presented as the mean ± SEM ( n = 4). (E,F) naïve CD4 + T cells were transduced with miR-124 inhibitor (+) or inhibitor control (–) before Th17-polarization using immobilized anti-CD3. CD126 expression and (p)-STAT3 activity were checked by western blots. Data are presented as the mean ± SEM. NS means no significance, * P
    Flow Cytometry Analysis Antibodies Against Cd4, supplied by BioLegend, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry analysis antibodies against cd4/product/BioLegend
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    89
    BioLegend flow cytometry blood
    Characterization of cell surface markers of activation by flow <t>cytometry</t> of donor neutrophils from whole blood (WB) (n = 6) and transmigrated through the small-airways model for 14 hours towards airway supernatant (TM ASN) (n = 6–9). ( A ) CD66b, ( B ) CD63, ( C ) CD16, ( D ) CD181, ( E ) HNE, ( F ) CD88, ( G ) HLA-DR, ( H ) CXCR4 (CD184), ( I ) Arg-1, ( J ) PD-L1. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. Samples were compared using the Mann-Whitney U test. * p
    Flow Cytometry Blood, supplied by BioLegend, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry blood/product/BioLegend
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    flow cytometry blood - by Bioz Stars, 2020-09
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    92
    BioLegend flow cytometry analysis
    Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma (A and B) <t>Flow-cytometry-sorted</t> and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O 2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4 + , CD8 + , and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4 + , CD8 + , and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p
    Flow Cytometry Analysis, supplied by BioLegend, used in various techniques. Bioz Stars score: 92/100, based on 226 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Phenotypic analysis of natural killer (NK) and Hodgkin lymphoma (HL) cells. a NK-92, haNK, and primary NK cells were tested for CD16 expression by flow cytometry. Gating for CD56-positive cells and CD16 expression on the each of the NK cells was compared to a murine isotype control. Additionally, HL cell lines (KM-H2, L-428, and L-540), NK cells (NK-92, haNK, and primary NK cells), and the K562 control cell line were tested for cell surface CD123. b Results of the HL cell lines as (%) expression. c Relative variation of the median fluorescence intensity (RMFI) to a murine isotype control in all cells

    Journal: Blood Cancer Journal

    Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

    doi: 10.1038/s41408-018-0168-2

    Figure Lengend Snippet: Phenotypic analysis of natural killer (NK) and Hodgkin lymphoma (HL) cells. a NK-92, haNK, and primary NK cells were tested for CD16 expression by flow cytometry. Gating for CD56-positive cells and CD16 expression on the each of the NK cells was compared to a murine isotype control. Additionally, HL cell lines (KM-H2, L-428, and L-540), NK cells (NK-92, haNK, and primary NK cells), and the K562 control cell line were tested for cell surface CD123. b Results of the HL cell lines as (%) expression. c Relative variation of the median fluorescence intensity (RMFI) to a murine isotype control in all cells

    Article Snippet: CD107a expression was analyzed using cell surface flow cytometry after a 4-h incubation period in the presence of the transport inhibitor Monensin (Biolegend), either for NK cells alone or in the presence of target cells (E:T of 10:1) with or without CSL362 (1 µg/mL).

    Techniques: Expressing, Flow Cytometry, Cytometry, Fluorescence

    Cytotoxicity of Hodgkin lymphoma (HL) and CSL362-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). a NK-92 dose-dependent killing efficacy of 3 HL cell lines (KM-H2, L-428, and L-540) using the 4-h standard chromium release assay (CRA). b At 5:1 effector-to-target (E:T) ratio, the killing efficacy of haNK cells against the three HL cell lines and the K562 control cell line is compared. c Analysis of CSL362-binding capacity to CD123-positive HL cells and to CD123-negative control cells. Using flow cytometry, a secondary fluorescein isothiocyanate-conjugated anti-human Fc monoclonal antibody (mAb) was added after preincubation of HL cells with increasing doses of CSL362. d In a 4-h CRA, CSL362-mediated ADCC was studied using haNK cells against CD123-positive HL cells at a 5:1 E:T ratio (KM-H2 and L-428). Negative ADCC controls. e CD123-negative target cells. f The murine-Fc anti-CD123 mAb (7G3) was used instead of CSL362 and NK-92 cells (CD16-negative) as effector cells

    Journal: Blood Cancer Journal

    Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

    doi: 10.1038/s41408-018-0168-2

    Figure Lengend Snippet: Cytotoxicity of Hodgkin lymphoma (HL) and CSL362-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). a NK-92 dose-dependent killing efficacy of 3 HL cell lines (KM-H2, L-428, and L-540) using the 4-h standard chromium release assay (CRA). b At 5:1 effector-to-target (E:T) ratio, the killing efficacy of haNK cells against the three HL cell lines and the K562 control cell line is compared. c Analysis of CSL362-binding capacity to CD123-positive HL cells and to CD123-negative control cells. Using flow cytometry, a secondary fluorescein isothiocyanate-conjugated anti-human Fc monoclonal antibody (mAb) was added after preincubation of HL cells with increasing doses of CSL362. d In a 4-h CRA, CSL362-mediated ADCC was studied using haNK cells against CD123-positive HL cells at a 5:1 E:T ratio (KM-H2 and L-428). Negative ADCC controls. e CD123-negative target cells. f The murine-Fc anti-CD123 mAb (7G3) was used instead of CSL362 and NK-92 cells (CD16-negative) as effector cells

    Article Snippet: CD107a expression was analyzed using cell surface flow cytometry after a 4-h incubation period in the presence of the transport inhibitor Monensin (Biolegend), either for NK cells alone or in the presence of target cells (E:T of 10:1) with or without CSL362 (1 µg/mL).

    Techniques: Release Assay, Binding Assay, Negative Control, Flow Cytometry, Cytometry

    Degranulation markers and cytokines. a haNK cells and target cells were co-cultured at 5:1 effector-to-target (E:T) ratio for 4 h with and without CSL362 (1 µg/mL). Using flow cytometry and gating for CD56+ cells, the dot plot expression of the degranulation marker CD107a vs standard side scatter is shown. b Cumulative expression of CD107a compared to the CD123-negative control cell line K562. The presence of interferon-γ ( c ) and tumor necrosis factor-α ( d ) was estimated by comparing the relative variation of the median fluorescence intensity (RMFI) of haNK cells in co-culture with L-428 cells (5:1 E:T ratio) in the presence or absence of CSL362 (1 µg/mL), to that of untreated haNK cells alone

    Journal: Blood Cancer Journal

    Article Title: Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1

    doi: 10.1038/s41408-018-0168-2

    Figure Lengend Snippet: Degranulation markers and cytokines. a haNK cells and target cells were co-cultured at 5:1 effector-to-target (E:T) ratio for 4 h with and without CSL362 (1 µg/mL). Using flow cytometry and gating for CD56+ cells, the dot plot expression of the degranulation marker CD107a vs standard side scatter is shown. b Cumulative expression of CD107a compared to the CD123-negative control cell line K562. The presence of interferon-γ ( c ) and tumor necrosis factor-α ( d ) was estimated by comparing the relative variation of the median fluorescence intensity (RMFI) of haNK cells in co-culture with L-428 cells (5:1 E:T ratio) in the presence or absence of CSL362 (1 µg/mL), to that of untreated haNK cells alone

    Article Snippet: CD107a expression was analyzed using cell surface flow cytometry after a 4-h incubation period in the presence of the transport inhibitor Monensin (Biolegend), either for NK cells alone or in the presence of target cells (E:T of 10:1) with or without CSL362 (1 µg/mL).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Expressing, Marker, Negative Control, Fluorescence, Co-Culture Assay

    VD restrained Th17 cells differentiation through miR-124 mediated inhibition of IL-6 signaling. CD4 + CD62L + cells from C57BL/6J were cultured in the Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 with or without VD. In some experiments, naïve CD4 + T cells were transduced with 10 nM miR-124 inhibitor for 24 h using Lipofectamine® 3000 before polarized into Th17 cells. (A) qRT-PCR showed that VD upregulates miR-124 expression in Th17 cells (72 h). (B–D) naïve CD4 + T cells were transduced with miR-124 inhibitor (i) or inhibitor control (iNC) before Th17-polarization using immobilized anti-CD3 or APCs system and flow cytometry was used to confirm IL-17 expression at protein level. Data are presented as the mean ± SEM ( n = 4). (E,F) naïve CD4 + T cells were transduced with miR-124 inhibitor (+) or inhibitor control (–) before Th17-polarization using immobilized anti-CD3. CD126 expression and (p)-STAT3 activity were checked by western blots. Data are presented as the mean ± SEM. NS means no significance, * P

    Journal: Frontiers in Immunology

    Article Title: 1,25-Dihydroxyvitamin D3 Ameliorates Collagen-Induced Arthritis via Suppression of Th17 Cells Through miR-124 Mediated Inhibition of IL-6 Signaling

    doi: 10.3389/fimmu.2019.00178

    Figure Lengend Snippet: VD restrained Th17 cells differentiation through miR-124 mediated inhibition of IL-6 signaling. CD4 + CD62L + cells from C57BL/6J were cultured in the Th17-polarizing conditions with immobilized anti-CD3 and soluble anti-CD28 with or without VD. In some experiments, naïve CD4 + T cells were transduced with 10 nM miR-124 inhibitor for 24 h using Lipofectamine® 3000 before polarized into Th17 cells. (A) qRT-PCR showed that VD upregulates miR-124 expression in Th17 cells (72 h). (B–D) naïve CD4 + T cells were transduced with miR-124 inhibitor (i) or inhibitor control (iNC) before Th17-polarization using immobilized anti-CD3 or APCs system and flow cytometry was used to confirm IL-17 expression at protein level. Data are presented as the mean ± SEM ( n = 4). (E,F) naïve CD4 + T cells were transduced with miR-124 inhibitor (+) or inhibitor control (–) before Th17-polarization using immobilized anti-CD3. CD126 expression and (p)-STAT3 activity were checked by western blots. Data are presented as the mean ± SEM. NS means no significance, * P

    Article Snippet: Flow Cytometry Analysis Antibodies against CD4 (GK1.5, PerCP/Cy5.5), IFN-γ (XMG1.2, APC), IL-17 (TC11-18H10.1, PE), Nrp-1 (Neuropilin-1, 3E12, PE) and CD126 (IL-6R α chain, D7715A7, APC) were from Biolegend.

    Techniques: Inhibition, Cell Culture, Transduction, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Activity Assay, Western Blot

    Characterization of cell surface markers of activation by flow cytometry of donor neutrophils from whole blood (WB) (n = 6) and transmigrated through the small-airways model for 14 hours towards airway supernatant (TM ASN) (n = 6–9). ( A ) CD66b, ( B ) CD63, ( C ) CD16, ( D ) CD181, ( E ) HNE, ( F ) CD88, ( G ) HLA-DR, ( H ) CXCR4 (CD184), ( I ) Arg-1, ( J ) PD-L1. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. Samples were compared using the Mann-Whitney U test. * p

    Journal: Scientific Reports

    Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

    doi: 10.1038/s41598-019-39726-w

    Figure Lengend Snippet: Characterization of cell surface markers of activation by flow cytometry of donor neutrophils from whole blood (WB) (n = 6) and transmigrated through the small-airways model for 14 hours towards airway supernatant (TM ASN) (n = 6–9). ( A ) CD66b, ( B ) CD63, ( C ) CD16, ( D ) CD181, ( E ) HNE, ( F ) CD88, ( G ) HLA-DR, ( H ) CXCR4 (CD184), ( I ) Arg-1, ( J ) PD-L1. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. Samples were compared using the Mann-Whitney U test. * p

    Article Snippet: Cell staining and flow cytometry Blood and airway neutrophils collected from patients in vivo and neutrophils transmigrated in the in vitro model were preincubated with human TruStain FcX receptor blocking solution (BioLegend, San Diego, CA) and Live/Dead Aqua (Thermo Fisher Scientific, Waltham, MA) for 10 min on ice in the dark followed by labeling antibodies listed in Table , for 30 min. All panels were applied to blood samples.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Western Blot, MANN-WHITNEY

    Characterization of blood (n = 8) and airway (n = 18) neutrophil cell surface markers of activation by flow cytometry. Samples were collected within 24 hours of endotracheal intubation (Day 1). Surface expression of integrins ( A ) CD11b, ( B ) CD11c, ( C ) CD49d, and ( D ) CD54, the immunoglobulin G receptor ( E ) CD32, ( F ) MHC II receptor, HLA-DR, and complement receptors ( G ) CD35 (C3b/C4b, CR1), and ( H ) CD88 (C5aR), and of the IL8 receptors ( I) CD181 and ( J ) CD182. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. * p

    Journal: Scientific Reports

    Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

    doi: 10.1038/s41598-019-39726-w

    Figure Lengend Snippet: Characterization of blood (n = 8) and airway (n = 18) neutrophil cell surface markers of activation by flow cytometry. Samples were collected within 24 hours of endotracheal intubation (Day 1). Surface expression of integrins ( A ) CD11b, ( B ) CD11c, ( C ) CD49d, and ( D ) CD54, the immunoglobulin G receptor ( E ) CD32, ( F ) MHC II receptor, HLA-DR, and complement receptors ( G ) CD35 (C3b/C4b, CR1), and ( H ) CD88 (C5aR), and of the IL8 receptors ( I) CD181 and ( J ) CD182. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. * p

    Article Snippet: Cell staining and flow cytometry Blood and airway neutrophils collected from patients in vivo and neutrophils transmigrated in the in vitro model were preincubated with human TruStain FcX receptor blocking solution (BioLegend, San Diego, CA) and Live/Dead Aqua (Thermo Fisher Scientific, Waltham, MA) for 10 min on ice in the dark followed by labeling antibodies listed in Table , for 30 min. All panels were applied to blood samples.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing

    Transmigration through the airway model primes healthy donor neutrophils to activate the respiratory burst when stimulated by N- formylmethionyl-leucine-phenylalanine (fMLF) as measured by dihydrorhodamine (DHR) flow cytometry assay. ( A ) Representative flow cytometry histograms depicting healthy donor neutrophil DHR response to fMLF without (blue histogram) and with priming by GM-CSF (100 ng/mL; green histogram) for 30 minutes at 37 °C, 5% CO 2 . PMA-stimulated (100 pg/mL) neutrophils serve as a positive control (orange histogram). Unstimulated, DHR loaded neutrophils are shown as a negation control (red histogram). ( B ) Representative flow cytometry data depicting the DHR fluorescence to fMLF stimulus for transmigrated to LTB4 (red histograms), transmigrated to pooled Day 1 ASN (blue histograms) and neutrophils co-incubated with pooled Day 1 ASN (green histograms) for one donor. PMA (magenta histogram) is included as a maximal respiratory burst positive control. ( C ) Change in the DHR response to fMLF stimulation for neutrophils allowed to transmigrate for 14 hours toward LTB4, ASN with no bacterial coinfection (low HNE), or ASN with bacterial coinfection (high HNE) diluted 1:3 in serum-free media. Data are reported as the mean and standard deviation for three donors and analyzed using ANOVA with a post-hoc Tukey test for multiple comparisons. * p

    Journal: Scientific Reports

    Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

    doi: 10.1038/s41598-019-39726-w

    Figure Lengend Snippet: Transmigration through the airway model primes healthy donor neutrophils to activate the respiratory burst when stimulated by N- formylmethionyl-leucine-phenylalanine (fMLF) as measured by dihydrorhodamine (DHR) flow cytometry assay. ( A ) Representative flow cytometry histograms depicting healthy donor neutrophil DHR response to fMLF without (blue histogram) and with priming by GM-CSF (100 ng/mL; green histogram) for 30 minutes at 37 °C, 5% CO 2 . PMA-stimulated (100 pg/mL) neutrophils serve as a positive control (orange histogram). Unstimulated, DHR loaded neutrophils are shown as a negation control (red histogram). ( B ) Representative flow cytometry data depicting the DHR fluorescence to fMLF stimulus for transmigrated to LTB4 (red histograms), transmigrated to pooled Day 1 ASN (blue histograms) and neutrophils co-incubated with pooled Day 1 ASN (green histograms) for one donor. PMA (magenta histogram) is included as a maximal respiratory burst positive control. ( C ) Change in the DHR response to fMLF stimulation for neutrophils allowed to transmigrate for 14 hours toward LTB4, ASN with no bacterial coinfection (low HNE), or ASN with bacterial coinfection (high HNE) diluted 1:3 in serum-free media. Data are reported as the mean and standard deviation for three donors and analyzed using ANOVA with a post-hoc Tukey test for multiple comparisons. * p

    Article Snippet: Cell staining and flow cytometry Blood and airway neutrophils collected from patients in vivo and neutrophils transmigrated in the in vitro model were preincubated with human TruStain FcX receptor blocking solution (BioLegend, San Diego, CA) and Live/Dead Aqua (Thermo Fisher Scientific, Waltham, MA) for 10 min on ice in the dark followed by labeling antibodies listed in Table , for 30 min. All panels were applied to blood samples.

    Techniques: Transmigration Assay, Flow Cytometry, Cytometry, Positive Control, Fluorescence, Incubation, Standard Deviation

    Characterization of blood and airway neutrophils markers by flow cytometry and markers of neutrophil activation in the plasma and airway fluid. Box plots of neutrophil cell surface markers for ( A ) CD62L, ( B) CD16, ( C ) CD66b, and ( D) CD63 in the blood (n = 8) and airway (n = 18) collected within 24 hours of endotracheal intubation (Day 1). Examples of histograms of the primary flow cytometry data are shown beside the box plot for each marker. Red and blue histograms represent blood and airway neutrophils, respectively. ( E ) Human neutrophil elastase activity assay and ( F ) matrix metalloproteinase 9 (MMP-9) protein levels from Day 1 plasma and cell-free airway fluid from tracheal aspirate samples. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. * p

    Journal: Scientific Reports

    Article Title: Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

    doi: 10.1038/s41598-019-39726-w

    Figure Lengend Snippet: Characterization of blood and airway neutrophils markers by flow cytometry and markers of neutrophil activation in the plasma and airway fluid. Box plots of neutrophil cell surface markers for ( A ) CD62L, ( B) CD16, ( C ) CD66b, and ( D) CD63 in the blood (n = 8) and airway (n = 18) collected within 24 hours of endotracheal intubation (Day 1). Examples of histograms of the primary flow cytometry data are shown beside the box plot for each marker. Red and blue histograms represent blood and airway neutrophils, respectively. ( E ) Human neutrophil elastase activity assay and ( F ) matrix metalloproteinase 9 (MMP-9) protein levels from Day 1 plasma and cell-free airway fluid from tracheal aspirate samples. Box plots depict median values, the box edges are the 25 th to 75 th interquartile ranges (IQR), and the whiskers are the 5−95% confidence intervals. * p

    Article Snippet: Cell staining and flow cytometry Blood and airway neutrophils collected from patients in vivo and neutrophils transmigrated in the in vitro model were preincubated with human TruStain FcX receptor blocking solution (BioLegend, San Diego, CA) and Live/Dead Aqua (Thermo Fisher Scientific, Waltham, MA) for 10 min on ice in the dark followed by labeling antibodies listed in Table , for 30 min. All panels were applied to blood samples.

    Techniques: Flow Cytometry, Cytometry, Activation Assay, Marker, Activity Assay

    Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma (A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O 2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4 + , CD8 + , and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4 + , CD8 + , and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

    Journal: Cell reports

    Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

    doi: 10.1016/j.celrep.2019.03.029

    Figure Lengend Snippet: Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma (A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O 2 overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4 + , CD8 + , and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4 + , CD8 + , and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

    Article Snippet: Flow cytometry analysis For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend.

    Techniques: Flow Cytometry, Cytometry, Mouse Assay, Expressing, Software

    In Vivo Treatment of Eto Causes a Survival Benefit in Immunocompetent Mice (A) Survival of WT or immunodeficient Rag 0/0 mice implanted with 4 × 10 5 GL-261 and treated with intracranial Eto administration beginning at day 7 after tumor implantation. (B) Quantification of Tregs and their ratios to other T cell subsets 48 hr after Eto treatment (day 9). (C) Survival of mice injected i.p. with 30 mg/kg Eto. (D) Flow-cytometric analysis of Treg infiltration and their ratios to other T cell subsets 48 h after second Eto treatment (9 days). Survival curves are from at least 7 mice per group from two independent experiments; statistical significance was calculated using log-rank analysis. Flow cytometry statistics were calculated and are indicated as percent positive population ± SEM; n = 5 per group, representative of two independent experiments in (B) and one experiment in (D). Unpaired t test analysis was used to calculate significance. *p

    Journal: Cell reports

    Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

    doi: 10.1016/j.celrep.2019.03.029

    Figure Lengend Snippet: In Vivo Treatment of Eto Causes a Survival Benefit in Immunocompetent Mice (A) Survival of WT or immunodeficient Rag 0/0 mice implanted with 4 × 10 5 GL-261 and treated with intracranial Eto administration beginning at day 7 after tumor implantation. (B) Quantification of Tregs and their ratios to other T cell subsets 48 hr after Eto treatment (day 9). (C) Survival of mice injected i.p. with 30 mg/kg Eto. (D) Flow-cytometric analysis of Treg infiltration and their ratios to other T cell subsets 48 h after second Eto treatment (9 days). Survival curves are from at least 7 mice per group from two independent experiments; statistical significance was calculated using log-rank analysis. Flow cytometry statistics were calculated and are indicated as percent positive population ± SEM; n = 5 per group, representative of two independent experiments in (B) and one experiment in (D). Unpaired t test analysis was used to calculate significance. *p

    Article Snippet: Flow cytometry analysis For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend.

    Techniques: In Vivo, Mouse Assay, Tumor Implantation, Injection, Flow Cytometry, Cytometry

    Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells (A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 μM) or the fatty acid uptake inhibitor SSO (200 μM), and the expression of various Tregs markers was assessed via flow cytometry. (B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 μM; B) or SSO (200 μM; E), and Foxp3 retention was determined. (C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry. (F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry. (H and I) Sorted and expanded WT or HIF-1α KO Tregs were pretreated with (H) Eto (200 μM) or (I) SSO (200 μM) for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, expansion indexes were determined via flow cytometry. Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

    Journal: Cell reports

    Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

    doi: 10.1016/j.celrep.2019.03.029

    Figure Lengend Snippet: Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells (A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 μM) or the fatty acid uptake inhibitor SSO (200 μM), and the expression of various Tregs markers was assessed via flow cytometry. (B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 μM; B) or SSO (200 μM; E), and Foxp3 retention was determined. (C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry. (F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry. (H and I) Sorted and expanded WT or HIF-1α KO Tregs were pretreated with (H) Eto (200 μM) or (I) SSO (200 μM) for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, expansion indexes were determined via flow cytometry. Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p

    Article Snippet: Flow cytometry analysis For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend.

    Techniques: Inhibition, Cell Culture, Expressing, Flow Cytometry, Cytometry, Co-Culture Assay

    HIF-1α KO Tregs Suppress CD8 + T Cell Proliferation Better than HIF-1α WT Tregs under Hypoxia due to Enhanced Glucose Oxidation (A and B) Sorted and expanded Tregs were plated with proliferation-dye-labeled CD8 + T cells at decreasing ratios to determine their suppressive capability under (A) 21% O 2 and (B) 1% O 2 . After 72 h, CD8 + T cell proliferation was analyzed. An n of 3 wells per ratio was analyzed, representative of three independent experiments. (C) Sorted and expanded Tregs were pre-treated with UK5099 (10 μm) or vehicle control for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, percent proliferation and expansion indexes were determined via flow cytometry. An n of 3 per condition was analyzed, from two independent experiments. (D) Sorted and expanded Tregs were pretreated with 1 mM DCA treatment overnight before suppressor assays were performed under 1% O 2 . Statistics were calculated as percent positive population ± SEM. Unpaired t test analysis was used to calculate significance. *p

    Journal: Cell reports

    Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

    doi: 10.1016/j.celrep.2019.03.029

    Figure Lengend Snippet: HIF-1α KO Tregs Suppress CD8 + T Cell Proliferation Better than HIF-1α WT Tregs under Hypoxia due to Enhanced Glucose Oxidation (A and B) Sorted and expanded Tregs were plated with proliferation-dye-labeled CD8 + T cells at decreasing ratios to determine their suppressive capability under (A) 21% O 2 and (B) 1% O 2 . After 72 h, CD8 + T cell proliferation was analyzed. An n of 3 wells per ratio was analyzed, representative of three independent experiments. (C) Sorted and expanded Tregs were pre-treated with UK5099 (10 μm) or vehicle control for 24 h before a suppressor assay was run under 1% O 2 . After 72 h, percent proliferation and expansion indexes were determined via flow cytometry. An n of 3 per condition was analyzed, from two independent experiments. (D) Sorted and expanded Tregs were pretreated with 1 mM DCA treatment overnight before suppressor assays were performed under 1% O 2 . Statistics were calculated as percent positive population ± SEM. Unpaired t test analysis was used to calculate significance. *p

    Article Snippet: Flow cytometry analysis For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend.

    Techniques: Labeling, Flow Cytometry, Cytometry

    Conditional Knockout of HIF-1α in Foxp3 + T Cells Inhibits Migration of Tregs to Brain Tumors In Vivo (A) To test Treg migration in vivo , splenic-sorted and expanded Tregs were co-labeled with eFluor 450 (HIF-1α WT) and eFluor 670 (HIF-1α KO) cell proliferation dyes and injected i.v. at a 1:1 ratio into mice harboring GL-261. (B) After 48 h, the brain, spleen, and DLN of mice injected with Tregs were isolated, and the ratio of control to HIF-1α KO Tregs was determined. (C) HIF-1α WT or HIF-1α KO mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and overall survival was determined. (D) After 2 weeks of tumor growth, Treg abundance was analyzed via flow cytometry. (E) Abundance of Foxp3 + cells from tumor-bearing mice was quantified in tissue sections. Kaplan-Meier curves are n = 7 per group from two independent experiments, and significance was calculated using log-rank analysis. Flow cytometry statistics shown as percent positive population ± SEM; n = 5 per group, representative of two experiments. In (B), the ratios of WT/HIF-1α KO Tregs were indicated as mean ± SEM. In (E), 3–5 fields per section were quantified for Foxp3 + DAPI + nuclei. n = 3 mice per group. Unpaired t test analysis was used to calculate significance. *p

    Journal: Cell reports

    Article Title: HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

    doi: 10.1016/j.celrep.2019.03.029

    Figure Lengend Snippet: Conditional Knockout of HIF-1α in Foxp3 + T Cells Inhibits Migration of Tregs to Brain Tumors In Vivo (A) To test Treg migration in vivo , splenic-sorted and expanded Tregs were co-labeled with eFluor 450 (HIF-1α WT) and eFluor 670 (HIF-1α KO) cell proliferation dyes and injected i.v. at a 1:1 ratio into mice harboring GL-261. (B) After 48 h, the brain, spleen, and DLN of mice injected with Tregs were isolated, and the ratio of control to HIF-1α KO Tregs was determined. (C) HIF-1α WT or HIF-1α KO mice were implanted with 4 × 10 5 GL-261 astrocytoma cells, and overall survival was determined. (D) After 2 weeks of tumor growth, Treg abundance was analyzed via flow cytometry. (E) Abundance of Foxp3 + cells from tumor-bearing mice was quantified in tissue sections. Kaplan-Meier curves are n = 7 per group from two independent experiments, and significance was calculated using log-rank analysis. Flow cytometry statistics shown as percent positive population ± SEM; n = 5 per group, representative of two experiments. In (B), the ratios of WT/HIF-1α KO Tregs were indicated as mean ± SEM. In (E), 3–5 fields per section were quantified for Foxp3 + DAPI + nuclei. n = 3 mice per group. Unpaired t test analysis was used to calculate significance. *p

    Article Snippet: Flow cytometry analysis For in-vitro studies the following flow cytometry panel was used in conjunction with CTV and viability dye: anti-CD3 Alexa700, CD4 PE-Cy7, CD8 APC, CD44 PE all at a 1:200 dilution purchased from Biolegend.

    Techniques: Knock-Out, Migration, In Vivo, Labeling, Injection, Mouse Assay, Isolation, Flow Cytometry, Cytometry