human peripheral blood mononuclear cells pbmcs  (GE Healthcare)

 
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    Structured Review

    GE Healthcare human peripheral blood mononuclear cells pbmcs
    Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 113 article reviews
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    Isolation:

    Article Title: Multiple Myeloma-Derived Extracellular Vesicles Induce Osteoclastogenesis through the Activation of the XBP1/IRE1α Axis
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque (GE Healthcare Bio Science, Uppsala, Sweden) separation technique. .. Preparation of Human Primary pOC and OCs PBMCs were cultured at 2.5 × 106 cells/mL α-Minimum Essential Media (MEM) supplemented with 10% FBS previously ultracentrifuged, 25 ng/mL of human recombinant RANK Ligand (Gibco, Life Technologies, Rockford, IL, USA), 25 ng/mL of human M-CSF (Gibco, Thermo Fisher Scientific, Rockford, IL, USA), and 10 nM dexamethasone (Sigma-Aldrich, Milano, Italy) (Human OC medium).

    Article Title: BxPC-3-Derived Small Extracellular Vesicles Induce FOXP3+ Treg through ATM-AMPK-Sirtuins-Mediated FOXOs Nuclear Translocations.
    Article Snippet: .. At Day 0, human peripheral blood mononuclear cells (PBMCs) from 32 age-matched healthy volunteers were isolated using Ficoll-Paque PLUS Medium (GE Healthcare) density gradient centrifugation. .. PBMCs were cultured with RPMI 1640 medium (GIBCO) at 37°C in a humidified atmosphere with 5% CO2.

    Article Title: Engagement of CD81 induces ezrin tyrosine phosphorylation and its cellular redistribution with filamentous actin
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMC) were isolated using Ficoll-Hypaque separation (Ficoll-Paque Plus; GE Healthcare Biosciences, Uppsala, Sweden), washed and suspended in RPMI containing 5% fetal calf serum. ..

    Article Title: N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFNγ-stimulated endothelial cells
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy adult volunteer donors by Ficoll-Hypaque density gradients (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). .. T-cells were purified using a Pan T-cell Isolation Kit (Miltenyi Biotec Inc., Auburn, CA, USA), according to the manufacturer’s instructions, seeded in 24 well plates (106 cells/ml in RPMI 1640/ 10% FBS (PAA Laboratories GmbH, Pasching, Austria)/ 1% penicillin and streptomycin (Sigma-Aldrich, St. Louis, USA) and stimulated for 3 days with 15 µl/ml Streptamer® anti-CD3/anti-CD28 Premix (IBA GmbH, Göttingen, Germany) in the presence or absence of 100 µM NOD (Novaliq GmbH, Heidelberg, Germany).

    Article Title: Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll-Paque Plus (GE Healthcare Bio-sciences, Uppsala, Sweden) density gradient centrifugation. .. PBMC were seeded at a density of 15 × 106 /well in 6-well plates and allowed to adhere for 1 h in serum-free RPMI (Lonza, Basel, Switzerland).

    Incubation:

    Article Title: Umbilical Cord-Derived Mesenchymal Stem Cells Are Able to Use bFGF Treatment and Represent a Superb Tool for Immunosuppressive Clinical Applications
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) were purified by a FicollTM Paque (GE Healthcare) density gradient, according to the manufacturer’s protocol, and incubated overnight at 37 °C in RPMI medium consisting of RPMI 1640 (Thermo Fisher Scientific), 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. .. The next day PBMC were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE, CFSE Cell Division Tracker Kit, BioLegend, CA, USA) and resuspended in RPMI medium to be plated at a concentration of 1.5 × 105 cells per well in a 96-well plate.

    Gradient Centrifugation:

    Article Title: BxPC-3-Derived Small Extracellular Vesicles Induce FOXP3+ Treg through ATM-AMPK-Sirtuins-Mediated FOXOs Nuclear Translocations.
    Article Snippet: .. At Day 0, human peripheral blood mononuclear cells (PBMCs) from 32 age-matched healthy volunteers were isolated using Ficoll-Paque PLUS Medium (GE Healthcare) density gradient centrifugation. .. PBMCs were cultured with RPMI 1640 medium (GIBCO) at 37°C in a humidified atmosphere with 5% CO2.

    Article Title: Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats by Ficoll-Paque Plus (GE Healthcare Bio-sciences, Uppsala, Sweden) density gradient centrifugation. .. PBMC were seeded at a density of 15 × 106 /well in 6-well plates and allowed to adhere for 1 h in serum-free RPMI (Lonza, Basel, Switzerland).

    Article Title: Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMC) from healthy volunteers were separated by density gradient centrifugation (Ficoll-Hypaque, Amersham, Biosciences) and the human fibroblasts from gingival tissue of healthy volunteers. .. PBMC and human fibroblasts were suspended in RPMI-1640 supplemented medium (10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.01 M Hepes) and incubated under humidified environment at 37°C and 5% CO2 .

    Centrifugation:

    Article Title: Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained by centrifugation at 700× g for 40 min at 20°C over a Ficoll-Isopaque (Amersham Biosciences, Freiburg, Germany) density gradient. .. After repeated washing (500× g for 10 min at 4°C) in phosphate-buffered saline (PBS) containing 0.3 mM EDTA, the monocytes were isolated by counterflow elutriation using the JE-6B elutriation system (Beckman Instruments, Palo Alto, CA, USA), as described previously [ ].

    Purification:

    Article Title: Umbilical Cord-Derived Mesenchymal Stem Cells Are Able to Use bFGF Treatment and Represent a Superb Tool for Immunosuppressive Clinical Applications
    Article Snippet: .. Human peripheral blood mononuclear cells (PBMCs) were purified by a FicollTM Paque (GE Healthcare) density gradient, according to the manufacturer’s protocol, and incubated overnight at 37 °C in RPMI medium consisting of RPMI 1640 (Thermo Fisher Scientific), 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. .. The next day PBMC were labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE, CFSE Cell Division Tracker Kit, BioLegend, CA, USA) and resuspended in RPMI medium to be plated at a concentration of 1.5 × 105 cells per well in a 96-well plate.

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    GE Healthcare flow cytometry labeling human peripheral blood mononuclear cells pbmcs
    Elevated expression of TAM receptors in monocytes of older adults <t>PBMCs</t> from younger and older adults were labeled for TAM receptors Tyro3, Axl, and Mer and expression was quantified by LSR-II flow <t>cytometry</t> as described [ 39 ]. Data shown is percent expression of TAM receptor in CD14 + , CD11c + monocytes, n = 16 young, N = 21 old, * indicates p
    Flow Cytometry Labeling Human Peripheral Blood Mononuclear Cells Pbmcs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare human pbmc
    Human CD8 + mucosa-associated invariant T (MAIT) cells mount a more intense interferon (IFN)-γ response to staphylococcal enterotoxin B (SEB) than do memory conventional T (T conv ) cells at the individual cell level. Human peripheral blood mononuclear cells <t>(PBMCs)</t> ( n = 7) were exposed to SEB for 24 h. IFN-γ-producing T cells were then immunophenotyped by flow cytometry to determine the percentages of Vα7.2 + CD161 high (MAIT) cells, Vα7.2 + CD161 - cells, CD45RO + CCR7 + Vα7.2 - central memory T (T CM ) cells, the CD4 + , CD8 + , or double negative (DN) subsets of CD45RO + CCR7 - Vα7.2 - effector memory T (T EM ) cells, and the CD4 + , CD8 + , or DN subsets of CD45RO - cells ( A ). The frequencies of bulk and IFN-γ + MAIT cells expressing CD4 and/or CD8 were also calculated and presented in a pie chart ( n = 8) ( B ). The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .
    Human Pbmc, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare human monocytes separation peripheral blood mononuclear cells
    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) <t>monocytes</t> and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive <t>cells</t> within freshly isolated <t>peripheral</t> <t>blood</t> <t>mononuclear</t> cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p
    Human Monocytes Separation Peripheral Blood Mononuclear Cells, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GE Healthcare human pbmc derived macrophages pbmc mφs
    Surface VTCN1 presentation is altered in APCs from NOD mice and T1D patients. A : Quantitative analysis ( left ) and representative images ( right ) of peritoneal macrophages (P.MΦ) stained for VTCN1 (red) and F4/80 (green). Data are reported as the mean RFU ± SEM ( n = 3–5 mice/group). Scale bars, 10 µm. D, diabetic; ND, nondiabetic. B : FACS of F4/80 + peritoneal cells from mice. Gray, isotype control. C : Representative images of BMDMs stained as in A . D : FACS of CD11c + DCs from pancreatic lymph nodes (PLN). E : Quantitative RT-PCR of VTCN1 mRNA in B6 g7 and ND-NOD P.MΦ. Data are reported as mean arbitrary units (AU) ± SEM ( n = 3 mice/group). F : VTCN1 immunoblot of medium conditioned by P.MΦ. Black line separates noncontiguous lanes from the same gel. G : ELISA of sVTCN1 in mouse sera. Data are reported as the mean ± SEM ( n = 12 mice/group). H : FACS ( left ) and immunohistochemistry ( right ) of human CD14 + <t>PBMC-MΦs.</t> cont.(P), nondiabetic parent of T1D-1 patient; gray, isotype control. Scale bars, 20 µm. I : Linear regression analysis of serum sVTCN1 and VTCN1 on PBMC-MΦs from humans. The overall correlation (solid black line) and individual correlations for each group (dashed lines of the corresponding color) are shown. J : Quantitative immunofluorescence analysis of VTCN1 on PBMC-MΦs from six T1D patients and five matching control subjects. Data are reported as the mean RFU ± SEM ( n > 100 cells/subject). Black lines show the mean for each group. Statistics were calculated by one-way ANOVA with Tukey post hoc test ( A and G ) and unpaired Student t test ( E ).
    Human Pbmc Derived Macrophages Pbmc Mφs, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Elevated expression of TAM receptors in monocytes of older adults PBMCs from younger and older adults were labeled for TAM receptors Tyro3, Axl, and Mer and expression was quantified by LSR-II flow cytometry as described [ 39 ]. Data shown is percent expression of TAM receptor in CD14 + , CD11c + monocytes, n = 16 young, N = 21 old, * indicates p

    Journal: Oncotarget

    Article Title: Age-related changes in expression and signaling of TAM receptor inflammatory regulators in monocytes

    doi: 10.18632/oncotarget.23851

    Figure Lengend Snippet: Elevated expression of TAM receptors in monocytes of older adults PBMCs from younger and older adults were labeled for TAM receptors Tyro3, Axl, and Mer and expression was quantified by LSR-II flow cytometry as described [ 39 ]. Data shown is percent expression of TAM receptor in CD14 + , CD11c + monocytes, n = 16 young, N = 21 old, * indicates p

    Article Snippet: Flow cytometry labeling Human peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood using Ficoll-Hypaque (GE Healthcare, NJ) as previously described [ ].

    Techniques: Expressing, Labeling, Flow Cytometry, Cytometry

    Human CD8 + mucosa-associated invariant T (MAIT) cells mount a more intense interferon (IFN)-γ response to staphylococcal enterotoxin B (SEB) than do memory conventional T (T conv ) cells at the individual cell level. Human peripheral blood mononuclear cells (PBMCs) ( n = 7) were exposed to SEB for 24 h. IFN-γ-producing T cells were then immunophenotyped by flow cytometry to determine the percentages of Vα7.2 + CD161 high (MAIT) cells, Vα7.2 + CD161 - cells, CD45RO + CCR7 + Vα7.2 - central memory T (T CM ) cells, the CD4 + , CD8 + , or double negative (DN) subsets of CD45RO + CCR7 - Vα7.2 - effector memory T (T EM ) cells, and the CD4 + , CD8 + , or DN subsets of CD45RO - cells ( A ). The frequencies of bulk and IFN-γ + MAIT cells expressing CD4 and/or CD8 were also calculated and presented in a pie chart ( n = 8) ( B ). The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Human CD8 + mucosa-associated invariant T (MAIT) cells mount a more intense interferon (IFN)-γ response to staphylococcal enterotoxin B (SEB) than do memory conventional T (T conv ) cells at the individual cell level. Human peripheral blood mononuclear cells (PBMCs) ( n = 7) were exposed to SEB for 24 h. IFN-γ-producing T cells were then immunophenotyped by flow cytometry to determine the percentages of Vα7.2 + CD161 high (MAIT) cells, Vα7.2 + CD161 - cells, CD45RO + CCR7 + Vα7.2 - central memory T (T CM ) cells, the CD4 + , CD8 + , or double negative (DN) subsets of CD45RO + CCR7 - Vα7.2 - effector memory T (T EM ) cells, and the CD4 + , CD8 + , or DN subsets of CD45RO - cells ( A ). The frequencies of bulk and IFN-γ + MAIT cells expressing CD4 and/or CD8 were also calculated and presented in a pie chart ( n = 8) ( B ). The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Expressing

    Mouse and human mucosa-associated invariant T (MAIT) cells can be activated by staphylococcal and streptococcal superantigens (SAgs) in a T cell receptor (TCR) Vβ-dependent manner. Mouse MAIT hybridoma lines 8D12, 6C2, and 17E6 were evaluated by flow cytometry for TCR Vβ8.1/2 expression ( A ). Indicated hybridoma(s) were exposed to 100 ng/mL of staphylococcal enterotoxin B (SEB) ( B-F ), SEB N23A ( B ), or several SAgs other than SEB ( F ) or stimulated with 0.5 μg/mL of an anti-CD3ε mAb ( C ) or with Klebsiella lysate ( E ) in the presence of DR4-transgenic (DR4 tg) bone marrow-derived dendritic cells (BMDCs) ( B-F ), wild-type C57BL/6 (B6) BMDCs ( B ), or γ-irradiated DR4 tg BMDCs ( D ). In several experiments, 5 μg/mL of an MHC-related protein 1 (MR1)-blocking monoclonal antibody (mAb) or an IgG2a isotype control was added to 8D12 cultures prior to stimulation with SEB or Klebsiella lysate ( E ). Culture supernatants were collected after 24 h, and interleukin (IL)-2 levels were quantified by ELISA. Representative data from 3 to 4 independent experiments yielding similar results are illustrated in panels A-F. Error bars represent SD to demonstrate variation among technical replicates. The frequencies of TCR Vβ13.2 + and Vβ2 + MAIT and conventional T (T conv ) cell subsets were determined among human peripheral blood mononuclear cells (PBMCs) isolated from 12 donors ( G ). To purify the above MAIT cell fractions, CD3 + Vα7.2 + CD161 + PBMCs were co-stained with monoclonal antibodies (mAbs) to TCR Vβ13.2 and Vβ2. MAIT cell subsets were then sorted and co-incubated with autologous CD14 + monocytes in the absence or presence of SEB ( n = 3 to 4). Twenty-four hours later, CD69 + cell percentages and interferon (IFN)-γ levels in culture supernatants were determined ( H ). Error bars in panels G and H represent SEM. **, ***, and **** indicate p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Mouse and human mucosa-associated invariant T (MAIT) cells can be activated by staphylococcal and streptococcal superantigens (SAgs) in a T cell receptor (TCR) Vβ-dependent manner. Mouse MAIT hybridoma lines 8D12, 6C2, and 17E6 were evaluated by flow cytometry for TCR Vβ8.1/2 expression ( A ). Indicated hybridoma(s) were exposed to 100 ng/mL of staphylococcal enterotoxin B (SEB) ( B-F ), SEB N23A ( B ), or several SAgs other than SEB ( F ) or stimulated with 0.5 μg/mL of an anti-CD3ε mAb ( C ) or with Klebsiella lysate ( E ) in the presence of DR4-transgenic (DR4 tg) bone marrow-derived dendritic cells (BMDCs) ( B-F ), wild-type C57BL/6 (B6) BMDCs ( B ), or γ-irradiated DR4 tg BMDCs ( D ). In several experiments, 5 μg/mL of an MHC-related protein 1 (MR1)-blocking monoclonal antibody (mAb) or an IgG2a isotype control was added to 8D12 cultures prior to stimulation with SEB or Klebsiella lysate ( E ). Culture supernatants were collected after 24 h, and interleukin (IL)-2 levels were quantified by ELISA. Representative data from 3 to 4 independent experiments yielding similar results are illustrated in panels A-F. Error bars represent SD to demonstrate variation among technical replicates. The frequencies of TCR Vβ13.2 + and Vβ2 + MAIT and conventional T (T conv ) cell subsets were determined among human peripheral blood mononuclear cells (PBMCs) isolated from 12 donors ( G ). To purify the above MAIT cell fractions, CD3 + Vα7.2 + CD161 + PBMCs were co-stained with monoclonal antibodies (mAbs) to TCR Vβ13.2 and Vβ2. MAIT cell subsets were then sorted and co-incubated with autologous CD14 + monocytes in the absence or presence of SEB ( n = 3 to 4). Twenty-four hours later, CD69 + cell percentages and interferon (IFN)-γ levels in culture supernatants were determined ( H ). Error bars in panels G and H represent SEM. **, ***, and **** indicate p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Expressing, Transgenic Assay, Derivative Assay, Irradiation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Incubation

    Staphylococcal enterotoxin B (SEB) stimulates human peripheral blood and hepatic mucosa-associated invariant T (MAIT) cells to produce classic pro-inflammatory cytokines except interleukin (IL)-17. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 100 ng/mL of SEB, and culture supernatants were collected at indicated time points for cytokine analysis ( A ). Data from 4 healthy donors were averaged and used to generate a heat map illustrating interferon (IFN)-γ, interleukin (IL)-2, IL-17A, and tumor necrosis factor (TNF)-α levels. PBMCs ( n = 8) were exposed to SEB, and the frequencies of IFN-γ + , IL-2 + , TNF-α + , and IL-17 + events among conventional T (T conv ) and MAIT cells were determined at indicated time points ( B ). Freshly isolated and SEB-stimulated PBMCs ( n = 3) were also examined to assess the intracellular T-bet and RORγT contents of MAIT cells relative to background staining with isotype controls (filled histograms in representative plots) ( C ). Non-parenchymal hepatic mononuclear cells (HMNCs) were isolated from tumor-free liver tissue samples of patients with colorectal carcinoma ( n = 20), in which CD3 + Vα7.2 + CD161 + MAIT cell frequencies were calculated and compared with those determined in 20 PBMC samples ( D ). HMNCs were incubated for 12 h or 24 h with SEB, followed by cytofluorimetric analysis of IFN-γ and IL-17 production by hepatic MAIT cells ( E ). In a limited number of experiments, blood MAIT and T conv cells were purified using a cell sorter and co-incubated with autologous CD14 + monocytes in the presence or absence of SEB. IFN-γ and IL-17A contents of culture supernatants were measured after 2 h, 6 h, or 12 h by ELISA ( F ). *, **, and **** denote p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Staphylococcal enterotoxin B (SEB) stimulates human peripheral blood and hepatic mucosa-associated invariant T (MAIT) cells to produce classic pro-inflammatory cytokines except interleukin (IL)-17. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 100 ng/mL of SEB, and culture supernatants were collected at indicated time points for cytokine analysis ( A ). Data from 4 healthy donors were averaged and used to generate a heat map illustrating interferon (IFN)-γ, interleukin (IL)-2, IL-17A, and tumor necrosis factor (TNF)-α levels. PBMCs ( n = 8) were exposed to SEB, and the frequencies of IFN-γ + , IL-2 + , TNF-α + , and IL-17 + events among conventional T (T conv ) and MAIT cells were determined at indicated time points ( B ). Freshly isolated and SEB-stimulated PBMCs ( n = 3) were also examined to assess the intracellular T-bet and RORγT contents of MAIT cells relative to background staining with isotype controls (filled histograms in representative plots) ( C ). Non-parenchymal hepatic mononuclear cells (HMNCs) were isolated from tumor-free liver tissue samples of patients with colorectal carcinoma ( n = 20), in which CD3 + Vα7.2 + CD161 + MAIT cell frequencies were calculated and compared with those determined in 20 PBMC samples ( D ). HMNCs were incubated for 12 h or 24 h with SEB, followed by cytofluorimetric analysis of IFN-γ and IL-17 production by hepatic MAIT cells ( E ). In a limited number of experiments, blood MAIT and T conv cells were purified using a cell sorter and co-incubated with autologous CD14 + monocytes in the presence or absence of SEB. IFN-γ and IL-17A contents of culture supernatants were measured after 2 h, 6 h, or 12 h by ELISA ( F ). *, **, and **** denote p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Isolation, Staining, Incubation, Purification, Enzyme-linked Immunosorbent Assay

    Staphylococcal enterotoxin B (SEB) stimulation of mucosa-associated invariant T (MAIT) cells results in lymphocyte-activation gene 3 (LAG-3)/ T cell immunoglobulin and mucin-3 (TIM-3) upregulation and interferes with their cognate antibacterial activity. Human peripheral blood mononuclear cells (PBMCs) ( n = 8) were left untreated or stimulated for 24 h with SEB. They were then washed and rested for 3 days before they were exposed to K . pneumoniae lysate. Interferon (IFN)-γ + , tumor necrosis factor (TNF)-α + , interleukin (IL)-2 + , and IL-17A + events among MAIT cells were enumerated 24 h later by intracellular cytokine staining ( A ). PBMCs from a separate cohort were subjected to SEB stimulation, followed by 24 h of resting, before they were challenged with either K . pneumoniae or E . coli lysate. Twenty-four hours later, cells were interrogated for their intracellular IFN-γ content ( n = 13) and evaluated for staining with Annexin V or Fixable Viability Dye ( n = 4) ( B ). SEB-exposed CD3 + cells co-expressing LAG-3 were divided into 5 subpopulations based on CD161 and Vα7.2 staining. The relative contribution of each subpopulation to total CD3/LAG-3 double-expressors is depicted in a pie chart generated using PBMCs from 8 donors ( C ). Concomitant upregulation of CD69 and LAG-3 by SEB, or lack thereof, was also examined in conventional T (T conv ) and MAIT cell compartments. Representative FACS plots are shown ( n = 8) ( D ). In additional experiments, the frequencies of LAG-3 + MAIT cells were determined in PBMC cultures containing 20 μM SB203580 or PD98059 ( E ), 5 ng/mL recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) ( F ), 5 μg/mL anti-IL-12 and anti-IL-18 ± 200 ng/mL cyclosporine A (CsA) ( n = 3) ( G ). The proportions of LAG-3 + TIM-3 - and LAG-3/TIM-3 double-expressors among T conv and MAIT cells were also calculated at indicated time points after SEB stimulation of PBMCs ( n = 8) ( H ). In separate experiments, PBMC cultures were stimulated for 24 h with SEB. Cells were washed, and cultures were replenished with fresh medium containing 20 μg/mL of an anti-human LAG-3 monoclonal antibody (mAb) or a mouse IgG1 isotype control. Klebsiella lysate was added to cultures, followed, 24 h later, by enumeration of IFN-γ + MAIT cells ( n = 3) ( I ). Error bars represent SEM. The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Staphylococcal enterotoxin B (SEB) stimulation of mucosa-associated invariant T (MAIT) cells results in lymphocyte-activation gene 3 (LAG-3)/ T cell immunoglobulin and mucin-3 (TIM-3) upregulation and interferes with their cognate antibacterial activity. Human peripheral blood mononuclear cells (PBMCs) ( n = 8) were left untreated or stimulated for 24 h with SEB. They were then washed and rested for 3 days before they were exposed to K . pneumoniae lysate. Interferon (IFN)-γ + , tumor necrosis factor (TNF)-α + , interleukin (IL)-2 + , and IL-17A + events among MAIT cells were enumerated 24 h later by intracellular cytokine staining ( A ). PBMCs from a separate cohort were subjected to SEB stimulation, followed by 24 h of resting, before they were challenged with either K . pneumoniae or E . coli lysate. Twenty-four hours later, cells were interrogated for their intracellular IFN-γ content ( n = 13) and evaluated for staining with Annexin V or Fixable Viability Dye ( n = 4) ( B ). SEB-exposed CD3 + cells co-expressing LAG-3 were divided into 5 subpopulations based on CD161 and Vα7.2 staining. The relative contribution of each subpopulation to total CD3/LAG-3 double-expressors is depicted in a pie chart generated using PBMCs from 8 donors ( C ). Concomitant upregulation of CD69 and LAG-3 by SEB, or lack thereof, was also examined in conventional T (T conv ) and MAIT cell compartments. Representative FACS plots are shown ( n = 8) ( D ). In additional experiments, the frequencies of LAG-3 + MAIT cells were determined in PBMC cultures containing 20 μM SB203580 or PD98059 ( E ), 5 ng/mL recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) ( F ), 5 μg/mL anti-IL-12 and anti-IL-18 ± 200 ng/mL cyclosporine A (CsA) ( n = 3) ( G ). The proportions of LAG-3 + TIM-3 - and LAG-3/TIM-3 double-expressors among T conv and MAIT cells were also calculated at indicated time points after SEB stimulation of PBMCs ( n = 8) ( H ). In separate experiments, PBMC cultures were stimulated for 24 h with SEB. Cells were washed, and cultures were replenished with fresh medium containing 20 μg/mL of an anti-human LAG-3 monoclonal antibody (mAb) or a mouse IgG1 isotype control. Klebsiella lysate was added to cultures, followed, 24 h later, by enumeration of IFN-γ + MAIT cells ( n = 3) ( I ). Error bars represent SEM. The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Activation Assay, Activity Assay, Staining, Expressing, Generated, FACS, Recombinant

    Staphylococcal enterotoxin B (SEB) can activate human mucosa-associated invariant T (MAIT) cells by an interleukin (IL)-12/IL-18-dependent mechanism. Peripheral blood mononuclear cells (PBMCs) were exposed to SEB for 24 h, and interferon (IFN)-γ + events were enumerated among Vβ13.2 + and Vβ2 + MAIT cells ( A ). Human PBMCs ( n = 4) were stimulated with SEB before culture supernatants were harvested at indicated time points to assay for IL-18, IL-12p70, IFN-α2, IL-7, and IL-15. Data were averaged to generate a heat map ( B ). The relative frequencies of CD218a + and CD218a - cells within the CD3 + IFN-γ + gate was determined at indicated time points post-SEB stimulation ( n = 3) ( C ). CD3 + cells exhibiting high, intermediate and low surface levels of CD218a were further analyzed for CD161 and Vα7.2 positivity ( D ). CD218a and CD212 expression by MAIT cells was assessed in untreated and SEB-stimulated PBMC cultures ( n = 7) ( E ). Filled and open histograms correspond to staining with isotype controls and anti-CD218a/CD212, respectively ( E ). In several experiments, neutralizing monoclonal antibodies (mAbs) to IFN-γ, IL-12, and/or IL-18 (or isotype control[s]) were added to PBMC cultures prior to SEB stimulation. Twenty-four hours later, the percentages of CD69 + ( F ) and cytokine + events ( G-H ) were determined among total ( F-H ) or fractionated ( H ) MAIT cells. In additional cultures, PBMCs were stimulated for 24 h with SEB or with recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) in parallel before cell-surface expression of CD69 ( n = 8) and intracellular IFN-γ accumulation ( n = 4) in MAIT cells were evaluated ( I ). Error bars represent SEM, and *** indicates a statistically significant difference with p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Staphylococcal enterotoxin B (SEB) can activate human mucosa-associated invariant T (MAIT) cells by an interleukin (IL)-12/IL-18-dependent mechanism. Peripheral blood mononuclear cells (PBMCs) were exposed to SEB for 24 h, and interferon (IFN)-γ + events were enumerated among Vβ13.2 + and Vβ2 + MAIT cells ( A ). Human PBMCs ( n = 4) were stimulated with SEB before culture supernatants were harvested at indicated time points to assay for IL-18, IL-12p70, IFN-α2, IL-7, and IL-15. Data were averaged to generate a heat map ( B ). The relative frequencies of CD218a + and CD218a - cells within the CD3 + IFN-γ + gate was determined at indicated time points post-SEB stimulation ( n = 3) ( C ). CD3 + cells exhibiting high, intermediate and low surface levels of CD218a were further analyzed for CD161 and Vα7.2 positivity ( D ). CD218a and CD212 expression by MAIT cells was assessed in untreated and SEB-stimulated PBMC cultures ( n = 7) ( E ). Filled and open histograms correspond to staining with isotype controls and anti-CD218a/CD212, respectively ( E ). In several experiments, neutralizing monoclonal antibodies (mAbs) to IFN-γ, IL-12, and/or IL-18 (or isotype control[s]) were added to PBMC cultures prior to SEB stimulation. Twenty-four hours later, the percentages of CD69 + ( F ) and cytokine + events ( G-H ) were determined among total ( F-H ) or fractionated ( H ) MAIT cells. In additional cultures, PBMCs were stimulated for 24 h with SEB or with recombinant human IL-12 (rIL-12) and/or recombinant human IL-18 (rIL-18) in parallel before cell-surface expression of CD69 ( n = 8) and intracellular IFN-γ accumulation ( n = 4) in MAIT cells were evaluated ( I ). Error bars represent SEM, and *** indicates a statistically significant difference with p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Expressing, Staining, Recombinant

    Rapid, exaggerated mucosa-associated invariant T (MAIT) cell responses to staphylococcal enterotoxin B (SEB) require the presence of HLA class II but not MHC-related protein 1 (MR1). Human peripheral blood mononuclear cells (PBMCs) were stimulated for 12 h with indicated doses of SEB ( A ) or for indicated durations with 100 ng/mL of SEB ( B ) before the frequency of CD69 + events and the mean fluorescence intensity (MFI) of CD69 were determined among MAIT and conventional T (T conv ) cells. Data were pooled from at least 3 independent experiments, and mean ± SEM values are shown ( n = 5 for A and n = 8 for B). PBMCs ( n = 9 in C; n = 3 in D) were incubated for 24 h with SEB ( C-D ) or Klebsiella lysate ( D ) in the presence of an HLA-DR-blocking monoclonal antibody (mAb) or an isotype control ( C ) or an anti-MR1 mAb ( D ). The percentage of interferon (IFN)-γ + events among MAIT cells is reported, and error bars represent SEM. The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Rapid, exaggerated mucosa-associated invariant T (MAIT) cell responses to staphylococcal enterotoxin B (SEB) require the presence of HLA class II but not MHC-related protein 1 (MR1). Human peripheral blood mononuclear cells (PBMCs) were stimulated for 12 h with indicated doses of SEB ( A ) or for indicated durations with 100 ng/mL of SEB ( B ) before the frequency of CD69 + events and the mean fluorescence intensity (MFI) of CD69 were determined among MAIT and conventional T (T conv ) cells. Data were pooled from at least 3 independent experiments, and mean ± SEM values are shown ( n = 5 for A and n = 8 for B). PBMCs ( n = 9 in C; n = 3 in D) were incubated for 24 h with SEB ( C-D ) or Klebsiella lysate ( D ) in the presence of an HLA-DR-blocking monoclonal antibody (mAb) or an isotype control ( C ) or an anti-MR1 mAb ( D ). The percentage of interferon (IFN)-γ + events among MAIT cells is reported, and error bars represent SEM. The underlying data for this figure can be found in S1 Data , and our gating strategies are provided in S2 Data .

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Fluorescence, Incubation, Blocking Assay

    Staphylococcal enterotoxin B (SEB) induces rapid expression of LAG-3, TIM-3 and PD-1 by human mucosa-associated invariant T (MAIT) cells in vivo. Human peripheral blood mononuclear cell—reconstituted NOD- scid  IL-2Rγ null  (hPBMC-NSG) mice ( n  = 3/group) were injected with PBS or 100 μg SEB. Twenty-four hours later, human interferon (IFN)-γ was measured in the serum by ELISA. Error bars represent SEM, and ** denotes  p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Staphylococcal enterotoxin B (SEB) induces rapid expression of LAG-3, TIM-3 and PD-1 by human mucosa-associated invariant T (MAIT) cells in vivo. Human peripheral blood mononuclear cell—reconstituted NOD- scid IL-2Rγ null (hPBMC-NSG) mice ( n = 3/group) were injected with PBS or 100 μg SEB. Twenty-four hours later, human interferon (IFN)-γ was measured in the serum by ELISA. Error bars represent SEM, and ** denotes p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Expressing, In Vivo, Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay

    IL-12/IL-18-mediated transactivation of mucosa-associated invariant T (MAIT) cells following staphylococcal enterotoxin B (SEB) stimulation requires signaling through p38 and MEK1/2. Human peripheral blood mononuclear cells (PBMCs) ( n = 6) were exposed to SEB in the absence or presence of 20 μM SB203580 and/or PD98059, and intracellular interferon (IFN)-γ accumulation in MAIT cells was detected after 24 h by flow cytometry ( A ). Intracellular phosphorylated p38 was traced in MAIT cells after stimulation with SEB in the absence or presence of anti-IL-12 and/or anti-IL-18 monoclonal antibodies (mAbs). Representative FACS plots are shown along with a bar graph summarizing data obtained from 3 individuals (*: p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: IL-12/IL-18-mediated transactivation of mucosa-associated invariant T (MAIT) cells following staphylococcal enterotoxin B (SEB) stimulation requires signaling through p38 and MEK1/2. Human peripheral blood mononuclear cells (PBMCs) ( n = 6) were exposed to SEB in the absence or presence of 20 μM SB203580 and/or PD98059, and intracellular interferon (IFN)-γ accumulation in MAIT cells was detected after 24 h by flow cytometry ( A ). Intracellular phosphorylated p38 was traced in MAIT cells after stimulation with SEB in the absence or presence of anti-IL-12 and/or anti-IL-18 monoclonal antibodies (mAbs). Representative FACS plots are shown along with a bar graph summarizing data obtained from 3 individuals (*: p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, FACS

    Human peripheral blood mucosa-associated invariant T (MAIT) cells are a predominant source of interferon (IFN)-γ after exposure to staphylococcal enterotoxin B (SEB). Human peripheral blood mononuclear cells (PBMCs) were left untreated or stimulated for 24 h with 100 ng/mL of SEB. Intracellular IFN-γ was detected by flow cytometry among bulk CD3 + , CD3 + CD161 - , CD3 + CD161 low , and CD3 + CD161 high cells ( A ). PBMCs were additionally stained with monoclonal antibodies (mAbs) to T cell receptor (TCR) γδ and TCR Vα7.2 and with PBS-57-loaded CD1d tetramer. Events corresponding to γδ T (green), invariant natural killer T ( i NKT) (red), MAIT (blue), and conventional T (T conv ) (cyan) cells were superimposed to generate a dot plot ( B ). The frequency of IFN-γ + cells and the mean fluorescence intensity (MFI) of IFN-γ staining were determined in 7 donors, each of whom is represented by a circle ( C ). Five subpopulations were defined among SEB-stimulated CD3 + cells based on their co-expression of CD161 and Vα7.2, or lack thereof. The proportion of IFN-γ + cells for each subpopulation is demonstrated in representative FACS plots ( D ). Historical data from 24 donors were subjected to Spearman’s rank correlation analysis to test the association between MAIT cell and IFN-γ + cell frequencies ( Fig 1E ). Mean ± SEM values are shown in C ( n = 7) and D ( n = 9). *, **, ***, and **** in panel C denote statistical differences with p

    Journal: PLoS Biology

    Article Title: MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

    doi: 10.1371/journal.pbio.2001930

    Figure Lengend Snippet: Human peripheral blood mucosa-associated invariant T (MAIT) cells are a predominant source of interferon (IFN)-γ after exposure to staphylococcal enterotoxin B (SEB). Human peripheral blood mononuclear cells (PBMCs) were left untreated or stimulated for 24 h with 100 ng/mL of SEB. Intracellular IFN-γ was detected by flow cytometry among bulk CD3 + , CD3 + CD161 - , CD3 + CD161 low , and CD3 + CD161 high cells ( A ). PBMCs were additionally stained with monoclonal antibodies (mAbs) to T cell receptor (TCR) γδ and TCR Vα7.2 and with PBS-57-loaded CD1d tetramer. Events corresponding to γδ T (green), invariant natural killer T ( i NKT) (red), MAIT (blue), and conventional T (T conv ) (cyan) cells were superimposed to generate a dot plot ( B ). The frequency of IFN-γ + cells and the mean fluorescence intensity (MFI) of IFN-γ staining were determined in 7 donors, each of whom is represented by a circle ( C ). Five subpopulations were defined among SEB-stimulated CD3 + cells based on their co-expression of CD161 and Vα7.2, or lack thereof. The proportion of IFN-γ + cells for each subpopulation is demonstrated in representative FACS plots ( D ). Historical data from 24 donors were subjected to Spearman’s rank correlation analysis to test the association between MAIT cell and IFN-γ + cell frequencies ( Fig 1E ). Mean ± SEM values are shown in C ( n = 7) and D ( n = 9). *, **, ***, and **** in panel C denote statistical differences with p

    Article Snippet: Human PBMC and HMNC isolation PBMCs were isolated from heparinized whole blood of healthy donors by density gradient centrifugation using low-endotoxin ( < 0.12 EU/mL) Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 50-mL SepMate tubes (Stemcell Technologies Inc., Vancouver, BC), as per manufacturer’s instructions.

    Techniques: Flow Cytometry, Cytometry, Staining, Fluorescence, Expressing, FACS

    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Article Snippet: Flow Cytometry and Human Monocytes Separation Peripheral blood mononuclear cells were isolated by a density gradient centrifugation over a Ficoll-Hypaque (Ficoll-Paque PLUS, GE Healthcare) from 20 ml of freshly venous blood from five healthy donors (HD), five relapsing MS patients (MS acute), and five remitting MS patients (MS stable).

    Techniques: Mass Spectrometry, In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

    Surface VTCN1 presentation is altered in APCs from NOD mice and T1D patients. A : Quantitative analysis ( left ) and representative images ( right ) of peritoneal macrophages (P.MΦ) stained for VTCN1 (red) and F4/80 (green). Data are reported as the mean RFU ± SEM ( n = 3–5 mice/group). Scale bars, 10 µm. D, diabetic; ND, nondiabetic. B : FACS of F4/80 + peritoneal cells from mice. Gray, isotype control. C : Representative images of BMDMs stained as in A . D : FACS of CD11c + DCs from pancreatic lymph nodes (PLN). E : Quantitative RT-PCR of VTCN1 mRNA in B6 g7 and ND-NOD P.MΦ. Data are reported as mean arbitrary units (AU) ± SEM ( n = 3 mice/group). F : VTCN1 immunoblot of medium conditioned by P.MΦ. Black line separates noncontiguous lanes from the same gel. G : ELISA of sVTCN1 in mouse sera. Data are reported as the mean ± SEM ( n = 12 mice/group). H : FACS ( left ) and immunohistochemistry ( right ) of human CD14 + PBMC-MΦs. cont.(P), nondiabetic parent of T1D-1 patient; gray, isotype control. Scale bars, 20 µm. I : Linear regression analysis of serum sVTCN1 and VTCN1 on PBMC-MΦs from humans. The overall correlation (solid black line) and individual correlations for each group (dashed lines of the corresponding color) are shown. J : Quantitative immunofluorescence analysis of VTCN1 on PBMC-MΦs from six T1D patients and five matching control subjects. Data are reported as the mean RFU ± SEM ( n > 100 cells/subject). Black lines show the mean for each group. Statistics were calculated by one-way ANOVA with Tukey post hoc test ( A and G ) and unpaired Student t test ( E ).

    Journal: Diabetes

    Article Title: Nardilysin-Dependent Proteolysis of Cell-Associated VTCN1 (B7-H4) Marks Type 1 Diabetes Development

    doi: 10.2337/db14-0213

    Figure Lengend Snippet: Surface VTCN1 presentation is altered in APCs from NOD mice and T1D patients. A : Quantitative analysis ( left ) and representative images ( right ) of peritoneal macrophages (P.MΦ) stained for VTCN1 (red) and F4/80 (green). Data are reported as the mean RFU ± SEM ( n = 3–5 mice/group). Scale bars, 10 µm. D, diabetic; ND, nondiabetic. B : FACS of F4/80 + peritoneal cells from mice. Gray, isotype control. C : Representative images of BMDMs stained as in A . D : FACS of CD11c + DCs from pancreatic lymph nodes (PLN). E : Quantitative RT-PCR of VTCN1 mRNA in B6 g7 and ND-NOD P.MΦ. Data are reported as mean arbitrary units (AU) ± SEM ( n = 3 mice/group). F : VTCN1 immunoblot of medium conditioned by P.MΦ. Black line separates noncontiguous lanes from the same gel. G : ELISA of sVTCN1 in mouse sera. Data are reported as the mean ± SEM ( n = 12 mice/group). H : FACS ( left ) and immunohistochemistry ( right ) of human CD14 + PBMC-MΦs. cont.(P), nondiabetic parent of T1D-1 patient; gray, isotype control. Scale bars, 20 µm. I : Linear regression analysis of serum sVTCN1 and VTCN1 on PBMC-MΦs from humans. The overall correlation (solid black line) and individual correlations for each group (dashed lines of the corresponding color) are shown. J : Quantitative immunofluorescence analysis of VTCN1 on PBMC-MΦs from six T1D patients and five matching control subjects. Data are reported as the mean RFU ± SEM ( n > 100 cells/subject). Black lines show the mean for each group. Statistics were calculated by one-way ANOVA with Tukey post hoc test ( A and G ) and unpaired Student t test ( E ).

    Article Snippet: To obtain human PBMC-derived macrophages (PBMC-MΦs), leukocytes isolated from blood by Ficoll-Paque Premium (GE Healthcare) density centrifugation were adhesion enhanced and cultured for 10 days with 50 ng/mL macrophage colony-stimulating factor.

    Techniques: Mouse Assay, Staining, FACS, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Immunofluorescence