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Merck KGaA mononuclear cells mncs
Mononuclear Cells Mncs, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 5 article reviews
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mononuclear cells mncs - by Bioz Stars, 2020-09
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Centrifugation:

Article Title: Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells
Article Snippet: .. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany), and CD34+ cells enriched by magnetic beads were further sorted (2×) by using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). .. Non-viable cells were removed by propidium iodide staining.

Article Title: Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents
Article Snippet: .. In brief: mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany). .. CD34+ cells from the MNC fraction were enriched by using magnetic beads, followed by 2× sorting steps using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).

Magnetic Beads:

Article Title: Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells
Article Snippet: .. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany), and CD34+ cells enriched by magnetic beads were further sorted (2×) by using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). .. Non-viable cells were removed by propidium iodide staining.

Isolation:

Article Title: Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells
Article Snippet: .. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany), and CD34+ cells enriched by magnetic beads were further sorted (2×) by using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). .. Non-viable cells were removed by propidium iodide staining.

Article Title: Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents
Article Snippet: .. In brief: mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany). .. CD34+ cells from the MNC fraction were enriched by using magnetic beads, followed by 2× sorting steps using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).

Article Title: Quantifying Adhesion Mechanisms and Dynamics of Human Hematopoietic Stem and Progenitor Cells
Article Snippet: .. Briefly, mononuclear cells (MNCs) were isolated by density gradient centrifugation using the Ficoll–Hypaque technique (Merck KGaA, Darmstadt, Germany). .. CD34+ cells from the MNC fraction were enriched by labelling with magnetic microbeads and sorted twice using an affinity column with the AutoMACS system (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).

Small Interfering RNA:

Article Title: CSF1R inhibitors exhibit antitumor activity in acute myeloid leukemia by blocking paracrine signals from support cells
Article Snippet: .. Within each patient sample, a small-interfering RNA (siRNA) “hit” was identified if its cell viability was at least 2 standard deviations less than the mean computed across all siRNAs tested (z score ≤ −2)., To evaluate apoptosis, mononuclear cells (MNCs) were exposed to either GW-2580 or ARRY-382 at 10 µM, and apoptosis was measured after 24, 48, and 72 hours by Annexin V staining (Guava Nexin assay; Merck Millipore, Billerica, MA). ..

Affinity Column:

Article Title: Simple Physical Model Unravels Influences of Chemokine on Shape Deformation and Migration of Human Hematopoietic Stem Cells
Article Snippet: .. Mononuclear cells (MNCs) were isolated by density-gradient centrifugation (Merck KGaA, Darmstadt, Germany), and CD34+ cells enriched by magnetic beads were further sorted (2×) by using an AutoMACS affinity column (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). .. Non-viable cells were removed by propidium iodide staining.

Staining:

Article Title: CSF1R inhibitors exhibit antitumor activity in acute myeloid leukemia by blocking paracrine signals from support cells
Article Snippet: .. Within each patient sample, a small-interfering RNA (siRNA) “hit” was identified if its cell viability was at least 2 standard deviations less than the mean computed across all siRNAs tested (z score ≤ −2)., To evaluate apoptosis, mononuclear cells (MNCs) were exposed to either GW-2580 or ARRY-382 at 10 µM, and apoptosis was measured after 24, 48, and 72 hours by Annexin V staining (Guava Nexin assay; Merck Millipore, Billerica, MA). ..

Gradient Centrifugation:

Article Title: Quantifying Adhesion Mechanisms and Dynamics of Human Hematopoietic Stem and Progenitor Cells
Article Snippet: .. Briefly, mononuclear cells (MNCs) were isolated by density gradient centrifugation using the Ficoll–Hypaque technique (Merck KGaA, Darmstadt, Germany). .. CD34+ cells from the MNC fraction were enriched by labelling with magnetic microbeads and sorted twice using an affinity column with the AutoMACS system (all Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany).

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  • 90
    Merck KGaA human pbmc derived macrophages
    <t>CLEC5A</t> mediates enhanced proinflammatory cytokine and chemokine induction in human M-Mϕ after influenza virus infection. M-Mϕ differentiated from the <t>PBMC</t> of 8 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK HA,NA or VN HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ and viral titers in culture supernatants (log 10 TCID 50 /ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatant (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.
    Human Pbmc Derived Macrophages, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human pbmc derived macrophages/product/Merck KGaA
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    93
    Merck KGaA pbmcs
    Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8 + granulocytes. ( A ) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8 + granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. ( B ) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8 + granulocytes. <t>PMNs</t> and <t>PBMCs</t> were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.
    Pbmcs, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Merck KGaA cd3 stained pbmcs
    Suppressive activity of peficitinib on proinflammatory cytokine production by peripheral blood mononuclear cells Peripheral blood mononuclear cells isolated from patients with RA ( A, B, E, F ) or SSc ( C, D, G, H ) were stimulated with <t>anti-CD3/anti-CD28</t> antibodies for 24 h ( A–D ) or recombinant human IL-2 for 72 h ( E–H ) in the presence of the indicated concentration of peficitinib. Proinflammatory cytokine production in the culture supernatants were assessed by bead-based immunoassays. Cell viability was assessed with CellTiter-Glo ( B, D, F, H ). Data represents mean or mean with S.E.M. of three to five independent experiments using different donors. pefi: peficitinib.
    Cd3 Stained Pbmcs, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Merck KGaA ifn γ elispot assay 2 5 × 105 pbmcs
    B- and T-cell responses in pigs immunized with a single dose and different amounts of B 2 T. Time course of the specific antibody responses in sera collected on the indicated days pi. ( A ) Total anti peptide B IgG titers analyzed by ELISA. Each point depicts mean antibody titers (calculated as described in Methods) ± SD for each group of pigs ( n = 5). ( B ) Virus neutralization titers, VNT, expressed as the reciprocal log10 of the last serum dilution that neutralized 100 TCID50 of FMDV isolate O/UKG 11/01. Each symbol represents the value for an individual pig (numbering is included). Horizontal lines indicate the geometric mean for each animal group. In no case individual spontaneous reactivity was observed in the titers determined at day 0. Dotted lines denotes the assay detection limit. In ( A , B ) arrows point FMDV challenge (day 25 pi). ( C ) Specific T-cell responses measured by an ex vivo <t>IFN-γ</t> ELISPOT at days 15 pi. The frequency of FMDV-specific IFN-γ secreting cells was determined as detailed in Methods. Horizontal bars represent the mean frequencies of IFN-γ release spots of triplicates of peripheral blood mononuclear cells <t>(PBMCs)</t> from pigs stimulated in vitro with B 2 T (circles) or T (T3A) (squares) peptides (pig numbering is included).
    Ifn γ Elispot Assay 2 5 × 105 Pbmcs, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human M-Mϕ after influenza virus infection. M-Mϕ differentiated from the PBMC of 8 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK HA,NA or VN HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ and viral titers in culture supernatants (log 10 TCID 50 /ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatant (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Journal: Journal of Virology

    Article Title: CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

    doi: 10.1128/JVI.01813-16

    Figure Lengend Snippet: CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human M-Mϕ after influenza virus infection. M-Mϕ differentiated from the PBMC of 8 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK HA,NA or VN HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ and viral titers in culture supernatants (log 10 TCID 50 /ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatant (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Article Snippet: After 1 h of adsorption, the virus inoculum was removed and the cells were washed once with phosphate-buffered saline (PBS), followed by incubation with infection medium (RPMI supplemented with 2% FBS) for 24 h. For Syk inhibition and CLEC5A blocking assay, human PBMC-derived macrophages were pretreated with the indicated concentrations of Bay 61-3606 (Merck Millipore) or anti-human CLEC5A antagonistic antibody for 1 h before and throughout the infection process.

    Techniques: Infection, Transfection, FACS, Recombinant, MANN-WHITNEY

    CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human GM-Mϕ after influenza virus infection. GM-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK HA,NA or VN HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected GM-Mϕ and viral titers in culture supernatant (log 10 TCID 50 /ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Journal: Journal of Virology

    Article Title: CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

    doi: 10.1128/JVI.01813-16

    Figure Lengend Snippet: CLEC5A mediates enhanced proinflammatory cytokine and chemokine induction in human GM-Mϕ after influenza virus infection. GM-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA, followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. The macrophages were then infected with HK HA,NA or VN HA,NA recombinant viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected GM-Mϕ and viral titers in culture supernatant (log 10 TCID 50 /ml in MDCK cells) (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Article Snippet: After 1 h of adsorption, the virus inoculum was removed and the cells were washed once with phosphate-buffered saline (PBS), followed by incubation with infection medium (RPMI supplemented with 2% FBS) for 24 h. For Syk inhibition and CLEC5A blocking assay, human PBMC-derived macrophages were pretreated with the indicated concentrations of Bay 61-3606 (Merck Millipore) or anti-human CLEC5A antagonistic antibody for 1 h before and throughout the infection process.

    Techniques: Infection, Transfection, FACS, Recombinant, MANN-WHITNEY

    CLEC5A-mediated proinflammatory response in human M-Mϕ after infections with influenza viruses of H1N1, H5N1, and H7N9 subtypes. Human M-Mϕ differentiated from PBMC of 2 independent donors were infected with A/Hong Kong/54/98 (H1N1), A/Vietnam/1203/04 (H5N1), or A/Shanghai/2/13 (H7N9) influenza viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Journal: Journal of Virology

    Article Title: CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

    doi: 10.1128/JVI.01813-16

    Figure Lengend Snippet: CLEC5A-mediated proinflammatory response in human M-Mϕ after infections with influenza viruses of H1N1, H5N1, and H7N9 subtypes. Human M-Mϕ differentiated from PBMC of 2 independent donors were infected with A/Hong Kong/54/98 (H1N1), A/Vietnam/1203/04 (H5N1), or A/Shanghai/2/13 (H7N9) influenza viruses at an MOI of 2 for 24 h to determine viral M gene copy numbers in infected M-Mϕ (A) and proinflammatory cytokines and chemokines in culture supernatants (means ± SD, in pg/ml) (B). P values from Mann-Whitney tests are shown.

    Article Snippet: After 1 h of adsorption, the virus inoculum was removed and the cells were washed once with phosphate-buffered saline (PBS), followed by incubation with infection medium (RPMI supplemented with 2% FBS) for 24 h. For Syk inhibition and CLEC5A blocking assay, human PBMC-derived macrophages were pretreated with the indicated concentrations of Bay 61-3606 (Merck Millipore) or anti-human CLEC5A antagonistic antibody for 1 h before and throughout the infection process.

    Techniques: Infection, MANN-WHITNEY

    Blocking CLEC5A-mediated signaling with Syk inhibitor (Bay 61-3606) and CLEC5A antagonizing antibodies reduced inflammatory response in human M-Mϕ after influenza virus infection. (A) Human M-Mϕ differentiated from the PBMC of 3 independent donors were treated with dimethyl sulfoxide or the Syk inhibitor Bay 61-3606 prior to and throughout the course of infection with HK HA,NA or VN HA,NA virus at an MOI of 2 to determine the TNF-α and IP-10 concentrations (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (B) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with 1 μg/ml of murine IgG1 isotype control antibody (Iso ctrl) or clones of anti-human CLEC5A antibodies, which were previously shown to possess antagonizing activity against DV or JEV infections, prior to and throughout the course of infection with VN HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (C) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with increasing concentrations of murine IgG1 isotype control antibody or the anti-CLEC5A antibody (clone 8H8F5) prior to and throughout the course of infection with VN HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. ns, not significant. (D) M-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. M-Mϕ were infected with VN HA,NA or UV-inactivated VN HA,NA (UV-VN HA,NA ) at an MOI of 2. Viral M gene copy numbers and levels of TNF-α and IP-10 (means ± SD, in pg/ml) in culture supernatant were determined at 24 h postinfection. P values from Mann-Whitney tests are shown.

    Journal: Journal of Virology

    Article Title: CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo

    doi: 10.1128/JVI.01813-16

    Figure Lengend Snippet: Blocking CLEC5A-mediated signaling with Syk inhibitor (Bay 61-3606) and CLEC5A antagonizing antibodies reduced inflammatory response in human M-Mϕ after influenza virus infection. (A) Human M-Mϕ differentiated from the PBMC of 3 independent donors were treated with dimethyl sulfoxide or the Syk inhibitor Bay 61-3606 prior to and throughout the course of infection with HK HA,NA or VN HA,NA virus at an MOI of 2 to determine the TNF-α and IP-10 concentrations (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (B) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with 1 μg/ml of murine IgG1 isotype control antibody (Iso ctrl) or clones of anti-human CLEC5A antibodies, which were previously shown to possess antagonizing activity against DV or JEV infections, prior to and throughout the course of infection with VN HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. (C) Human M-Mϕ differentiated from the PBMC of 4 independent donors were incubated with increasing concentrations of murine IgG1 isotype control antibody or the anti-CLEC5A antibody (clone 8H8F5) prior to and throughout the course of infection with VN HA,NA virus at an MOI of 2 to determine the level of IP-10 (means ± SD, in pg/ml) from culture supernatants at 24 h postinfection. ns, not significant. (D) M-Mϕ differentiated from the PBMC of 2 independent donors were transfected with CLEC5A gene-specific or nontargeting siRNA followed by cell sorting to obtain the CLEC5A-positive (CLEC5A_pos) and CLEC5A-negative (CLEC5A_neg) populations. M-Mϕ were infected with VN HA,NA or UV-inactivated VN HA,NA (UV-VN HA,NA ) at an MOI of 2. Viral M gene copy numbers and levels of TNF-α and IP-10 (means ± SD, in pg/ml) in culture supernatant were determined at 24 h postinfection. P values from Mann-Whitney tests are shown.

    Article Snippet: After 1 h of adsorption, the virus inoculum was removed and the cells were washed once with phosphate-buffered saline (PBS), followed by incubation with infection medium (RPMI supplemented with 2% FBS) for 24 h. For Syk inhibition and CLEC5A blocking assay, human PBMC-derived macrophages were pretreated with the indicated concentrations of Bay 61-3606 (Merck Millipore) or anti-human CLEC5A antagonistic antibody for 1 h before and throughout the infection process.

    Techniques: Blocking Assay, Infection, Incubation, Clone Assay, Activity Assay, Transfection, FACS, MANN-WHITNEY

    Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8 + granulocytes. ( A ) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8 + granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. ( B ) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8 + granulocytes. PMNs and PBMCs were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Requirement of FcγRs, dynamism of plasma membrane, and actin recruitment in detection of CD8 + granulocytes. ( A ) Involvement of FcγRII (CD32) and FcγRIII (CD16) in the detection of CD8 + granulocytes. Heparinized blood samples were pre-incubated with the anti- FcγRII (CD32) Ab (AT10) or with the anti-FcγRIII (CD16) Ab (3G8). After the pre-incubation, the samples were made to react with the PE or PECy5-labeled anti-CD8α Ab (HIT8a). After depletion of erythrocytes, the cells were re-suspended in PBS followed by reaction with the FITC or PE-labeled anti-CD15 Ab (H198), and then served for FCM. PE or PECy5-labeled mouse IgG1 and FITC or PE-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. ( B ) Effects of plasma membrane fixation and inhibition of actin recruitment in the detection of CD8 + granulocytes. PMNs and PBMCs were mixed together. For fixation of the plasma membrane, the cells were exposed to 4% PFA for 10 min at room temperature. After washing 3 times with PBS, the cells were re-suspended in the autologous serum. The samples were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. For inhibition of actin recruitment, the mixture of PMNs and PBMCs was made to react with CyD (2 µg/ml) for 30 min at 37°C. After the pre-incubation, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 (H198), and then served for FCM. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively. These experiments were repeated 3 times.

    Article Snippet: Effects of complement inhibition and blockade of HAMA on FcγR-mediated trogocytosis First, PMNs (0.5×106 ) and PBMCs (0.5×106 ) were re-suspended in the autologous serum (100 µl) with or without 200 µg/ml of C1 inhibitor (Merck Millipore, Schwalbach, Germany).

    Techniques: Incubation, Labeling, Inhibition

    Requirement of serum and cell-to-cell interaction with T cells in detection of CD8 + granulocytes. ( A ) mRNA expressions of CD3ε, CD11b, CD8α, and CD8β in granulocytes (CD15 + PMNs) and T cells (CD3 + cells) determined by RT-PCR. These cells were separated from blood samples as described in Materials and Methods . The quality of RNA samples was verified by the expression of GAPDH. ( B ) Requirement of serum for detection of CD8 + granulocytes. PMNs and PBMCs separated from heparinized peripheral blood were mixed in PBS or autologous serum. These cells were incubated with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. ( C ) Requirement of cell-to-cell contact with T cells for detection of CD8 + granulocytes. PMNs and T cells were separated from heparinized blood samples. PMNs were cultured with or without T cells in the autologous serum. In the co-culture wells, PMNs were cultured separately from T cells using the transwell chambers or mixed together with T cells. Subsequently, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. These experiments were repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Requirement of serum and cell-to-cell interaction with T cells in detection of CD8 + granulocytes. ( A ) mRNA expressions of CD3ε, CD11b, CD8α, and CD8β in granulocytes (CD15 + PMNs) and T cells (CD3 + cells) determined by RT-PCR. These cells were separated from blood samples as described in Materials and Methods . The quality of RNA samples was verified by the expression of GAPDH. ( B ) Requirement of serum for detection of CD8 + granulocytes. PMNs and PBMCs separated from heparinized peripheral blood were mixed in PBS or autologous serum. These cells were incubated with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. ( C ) Requirement of cell-to-cell contact with T cells for detection of CD8 + granulocytes. PMNs and T cells were separated from heparinized blood samples. PMNs were cultured with or without T cells in the autologous serum. In the co-culture wells, PMNs were cultured separately from T cells using the transwell chambers or mixed together with T cells. Subsequently, the cells were made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. These experiments were repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Article Snippet: Effects of complement inhibition and blockade of HAMA on FcγR-mediated trogocytosis First, PMNs (0.5×106 ) and PBMCs (0.5×106 ) were re-suspended in the autologous serum (100 µl) with or without 200 µg/ml of C1 inhibitor (Merck Millipore, Schwalbach, Germany).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Labeling, Cell Culture, Co-Culture Assay

    Characterization of serum factors that contribute to FcγR-mediated trogocytosis. ( A ) Heat instability of serum factors that contribute to FcγR-mediated trogocytosis (n = 5). PMNs and PBMCs were mixed in sera, which had been heated at 56°C for 30 min, or sera without heat inactivation. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. Student t -test for paired samples was applied for statistical analysis. ( B ) Molecular weight range of serum factors that contribute to FcγR-mediated trogocytosis. PMNs and PBMCs were mixed in sera, which had been fractionated into those with molecular weight of more than 100 kDa or less than 100 kDa. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. This experiment was repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Journal: PLoS ONE

    Article Title: Mechanism of Fc? Receptor-Mediated Trogocytosis-Based False-Positive Results in Flow Cytometry

    doi: 10.1371/journal.pone.0052918

    Figure Lengend Snippet: Characterization of serum factors that contribute to FcγR-mediated trogocytosis. ( A ) Heat instability of serum factors that contribute to FcγR-mediated trogocytosis (n = 5). PMNs and PBMCs were mixed in sera, which had been heated at 56°C for 30 min, or sera without heat inactivation. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. Student t -test for paired samples was applied for statistical analysis. ( B ) Molecular weight range of serum factors that contribute to FcγR-mediated trogocytosis. PMNs and PBMCs were mixed in sera, which had been fractionated into those with molecular weight of more than 100 kDa or less than 100 kDa. These cells were then made to react with the PE-labeled anti-CD8α Ab (HIT8a). After removal of unbound Abs, the cells were re-suspended in PBS followed by reaction with the FITC-labeled anti-CD15 Ab (H198), and then served for FCM. This experiment was repeated 3 times. PE-labeled mouse IgG1 and FITC-labeled mouse IgM were used as isotype-matched controls for HIT8a and H198, respectively.

    Article Snippet: Effects of complement inhibition and blockade of HAMA on FcγR-mediated trogocytosis First, PMNs (0.5×106 ) and PBMCs (0.5×106 ) were re-suspended in the autologous serum (100 µl) with or without 200 µg/ml of C1 inhibitor (Merck Millipore, Schwalbach, Germany).

    Techniques: Labeling, Molecular Weight

    Suppressive activity of peficitinib on proinflammatory cytokine production by peripheral blood mononuclear cells Peripheral blood mononuclear cells isolated from patients with RA ( A, B, E, F ) or SSc ( C, D, G, H ) were stimulated with anti-CD3/anti-CD28 antibodies for 24 h ( A–D ) or recombinant human IL-2 for 72 h ( E–H ) in the presence of the indicated concentration of peficitinib. Proinflammatory cytokine production in the culture supernatants were assessed by bead-based immunoassays. Cell viability was assessed with CellTiter-Glo ( B, D, F, H ). Data represents mean or mean with S.E.M. of three to five independent experiments using different donors. pefi: peficitinib.

    Journal: Rheumatology (Oxford, England)

    Article Title: In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors

    doi: 10.1093/rheumatology/kez526

    Figure Lengend Snippet: Suppressive activity of peficitinib on proinflammatory cytokine production by peripheral blood mononuclear cells Peripheral blood mononuclear cells isolated from patients with RA ( A, B, E, F ) or SSc ( C, D, G, H ) were stimulated with anti-CD3/anti-CD28 antibodies for 24 h ( A–D ) or recombinant human IL-2 for 72 h ( E–H ) in the presence of the indicated concentration of peficitinib. Proinflammatory cytokine production in the culture supernatants were assessed by bead-based immunoassays. Cell viability was assessed with CellTiter-Glo ( B, D, F, H ). Data represents mean or mean with S.E.M. of three to five independent experiments using different donors. pefi: peficitinib.

    Article Snippet: CD3-stained PBMCs or serum-starved normal human dermal fibroblast cells (1.0 × 105 cells/sample) cultured in serum-free DMEM medium (Merck, Darmstadt, Germany) were pre-incubated with the test compounds at designated concentrations for 10 min at 37°C and treated with recombinant human cytokines for an additional 15 min.

    Techniques: Activity Assay, Isolation, Recombinant, Concentration Assay

    Constitutive activation of STATs in peripheral blood mononuclear cells from patients with RA and SSc Basal phosphorylated STATs level in CD3 + T-cells ( A ) and in monocytes ( B ). ( C ) Plasma cytokine levels were assessed by bead-based immunoassays. Samples with cytokine levels that were below the assay’s lower limit of detection were assigned the values of the midpoint between the lower limit of detection and zero. The horizontal bars in the figure indicate the means. Numbers in brackets on the abscissa represent the number of subjects in each group. Statistical analyses were performed using Dunn’s multiple comparisons test (HV vs SSc, HV vs RA). * P

    Journal: Rheumatology (Oxford, England)

    Article Title: In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors

    doi: 10.1093/rheumatology/kez526

    Figure Lengend Snippet: Constitutive activation of STATs in peripheral blood mononuclear cells from patients with RA and SSc Basal phosphorylated STATs level in CD3 + T-cells ( A ) and in monocytes ( B ). ( C ) Plasma cytokine levels were assessed by bead-based immunoassays. Samples with cytokine levels that were below the assay’s lower limit of detection were assigned the values of the midpoint between the lower limit of detection and zero. The horizontal bars in the figure indicate the means. Numbers in brackets on the abscissa represent the number of subjects in each group. Statistical analyses were performed using Dunn’s multiple comparisons test (HV vs SSc, HV vs RA). * P

    Article Snippet: CD3-stained PBMCs or serum-starved normal human dermal fibroblast cells (1.0 × 105 cells/sample) cultured in serum-free DMEM medium (Merck, Darmstadt, Germany) were pre-incubated with the test compounds at designated concentrations for 10 min at 37°C and treated with recombinant human cytokines for an additional 15 min.

    Techniques: Activation Assay

    B- and T-cell responses in pigs immunized with a single dose and different amounts of B 2 T. Time course of the specific antibody responses in sera collected on the indicated days pi. ( A ) Total anti peptide B IgG titers analyzed by ELISA. Each point depicts mean antibody titers (calculated as described in Methods) ± SD for each group of pigs ( n = 5). ( B ) Virus neutralization titers, VNT, expressed as the reciprocal log10 of the last serum dilution that neutralized 100 TCID50 of FMDV isolate O/UKG 11/01. Each symbol represents the value for an individual pig (numbering is included). Horizontal lines indicate the geometric mean for each animal group. In no case individual spontaneous reactivity was observed in the titers determined at day 0. Dotted lines denotes the assay detection limit. In ( A , B ) arrows point FMDV challenge (day 25 pi). ( C ) Specific T-cell responses measured by an ex vivo IFN-γ ELISPOT at days 15 pi. The frequency of FMDV-specific IFN-γ secreting cells was determined as detailed in Methods. Horizontal bars represent the mean frequencies of IFN-γ release spots of triplicates of peripheral blood mononuclear cells (PBMCs) from pigs stimulated in vitro with B 2 T (circles) or T (T3A) (squares) peptides (pig numbering is included).

    Journal: Vaccines

    Article Title: A Single Dose of Dendrimer B2T Peptide Vaccine Partially Protects Pigs against Foot-and-Mouth Disease Virus Infection

    doi: 10.3390/vaccines8010019

    Figure Lengend Snippet: B- and T-cell responses in pigs immunized with a single dose and different amounts of B 2 T. Time course of the specific antibody responses in sera collected on the indicated days pi. ( A ) Total anti peptide B IgG titers analyzed by ELISA. Each point depicts mean antibody titers (calculated as described in Methods) ± SD for each group of pigs ( n = 5). ( B ) Virus neutralization titers, VNT, expressed as the reciprocal log10 of the last serum dilution that neutralized 100 TCID50 of FMDV isolate O/UKG 11/01. Each symbol represents the value for an individual pig (numbering is included). Horizontal lines indicate the geometric mean for each animal group. In no case individual spontaneous reactivity was observed in the titers determined at day 0. Dotted lines denotes the assay detection limit. In ( A , B ) arrows point FMDV challenge (day 25 pi). ( C ) Specific T-cell responses measured by an ex vivo IFN-γ ELISPOT at days 15 pi. The frequency of FMDV-specific IFN-γ secreting cells was determined as detailed in Methods. Horizontal bars represent the mean frequencies of IFN-γ release spots of triplicates of peripheral blood mononuclear cells (PBMCs) from pigs stimulated in vitro with B 2 T (circles) or T (T3A) (squares) peptides (pig numbering is included).

    Article Snippet: For the IFN-γ ELISPOT assay 2.5 × 105 PBMCs were shed in triplicate wells of Immobilon-P plates (Merck Millipore, Burlington, MA, USA) coated with 5 µg/mL of anti-pig IFN-γ antibody (clone P2G10, Becton Dickinson, Franklin Lakes, NJ, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Ex Vivo, Enzyme-linked Immunospot, In Vitro