Structured Review

Millipore sterile phosphate buffered saline pbs
The mitogen-activated protein kinase pathway is essential for <t>LPS-induced</t> Fra-1 expression in macrophages. ( A ) MH-S cells were incubated with U0126, SP600125 (SP6), or SB202190 (SB) for 30 minutes and then treated with LPS for 6 hours. Fra-1 mRNA expression was analyzed by real-time polymerase chain reaction. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, <t>PBS</t> versus LPS; ( n = 3). † P ≤ 0.05, vehicle versus inhibitor. ( B ) MH-S cells were transfected with −213/+132 bp Fra-1 promoter (213-Luc) reporter construct. After 24 hours, cells were incubated with inhibitors as indicated for 30 minutes and then treated with LPS for 6 hours. The fold-activation of reporter activity by LPS was calculated using the values obtained from vehicle (PBS)-treated cells as 1.0. Data are presented as mean ± SEM ( n = 3). Experiment was repeated to obtain reproducible result.
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Images

1) Product Images from "c-Jun Is Required for Nuclear Factor-κB–Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages"

Article Title: c-Jun Is Required for Nuclear Factor-κB–Dependent, LPS-Stimulated Fos-Related Antigen-1 Transcription in Alveolar Macrophages

Journal: American Journal of Respiratory Cell and Molecular Biology

doi: 10.1165/rcmb.2016-0028OC

The mitogen-activated protein kinase pathway is essential for LPS-induced Fra-1 expression in macrophages. ( A ) MH-S cells were incubated with U0126, SP600125 (SP6), or SB202190 (SB) for 30 minutes and then treated with LPS for 6 hours. Fra-1 mRNA expression was analyzed by real-time polymerase chain reaction. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; ( n = 3). † P ≤ 0.05, vehicle versus inhibitor. ( B ) MH-S cells were transfected with −213/+132 bp Fra-1 promoter (213-Luc) reporter construct. After 24 hours, cells were incubated with inhibitors as indicated for 30 minutes and then treated with LPS for 6 hours. The fold-activation of reporter activity by LPS was calculated using the values obtained from vehicle (PBS)-treated cells as 1.0. Data are presented as mean ± SEM ( n = 3). Experiment was repeated to obtain reproducible result.
Figure Legend Snippet: The mitogen-activated protein kinase pathway is essential for LPS-induced Fra-1 expression in macrophages. ( A ) MH-S cells were incubated with U0126, SP600125 (SP6), or SB202190 (SB) for 30 minutes and then treated with LPS for 6 hours. Fra-1 mRNA expression was analyzed by real-time polymerase chain reaction. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; ( n = 3). † P ≤ 0.05, vehicle versus inhibitor. ( B ) MH-S cells were transfected with −213/+132 bp Fra-1 promoter (213-Luc) reporter construct. After 24 hours, cells were incubated with inhibitors as indicated for 30 minutes and then treated with LPS for 6 hours. The fold-activation of reporter activity by LPS was calculated using the values obtained from vehicle (PBS)-treated cells as 1.0. Data are presented as mean ± SEM ( n = 3). Experiment was repeated to obtain reproducible result.

Techniques Used: Expressing, Incubation, Real-time Polymerase Chain Reaction, Transfection, Construct, Activation Assay, Activity Assay

The effects of Fra-1 deficiency on LPS-stimulated NF-κB–dependent cytokine expression in lung alveolar macrophages in vivo . ( A ) Fra-1 +/+ and Fra-1 Δ/Δ mice were intratracheally (i.t.) instilled with PBS or LPS (10 μg/mouse) ( n = 4) and killed after 3 hours, and alveolar macrophages from their lungs were isolated as outlined in the schema A . Analysis of LPS-stimulated ( B ) proinflammatory and ( C ) antiinflammatory cytokine expression in alveolar macrophages of Fra-1 +/+ and Fra-1 Δ/Δ mice treated with LPS. ( D ) Fra-1 mRNA expression in alveolar macrophages of Fra-1 +/+ mice treated with LPS. Data are presented as mean ± SEM ( n = 3–4). * P
Figure Legend Snippet: The effects of Fra-1 deficiency on LPS-stimulated NF-κB–dependent cytokine expression in lung alveolar macrophages in vivo . ( A ) Fra-1 +/+ and Fra-1 Δ/Δ mice were intratracheally (i.t.) instilled with PBS or LPS (10 μg/mouse) ( n = 4) and killed after 3 hours, and alveolar macrophages from their lungs were isolated as outlined in the schema A . Analysis of LPS-stimulated ( B ) proinflammatory and ( C ) antiinflammatory cytokine expression in alveolar macrophages of Fra-1 +/+ and Fra-1 Δ/Δ mice treated with LPS. ( D ) Fra-1 mRNA expression in alveolar macrophages of Fra-1 +/+ mice treated with LPS. Data are presented as mean ± SEM ( n = 3–4). * P

Techniques Used: Expressing, In Vivo, Mouse Assay, Isolation

LPS induces Fra-1 expression in alveolar macrophages. ( A ) Cells were cultured with medium containing 2% FBS for 3 hours and then stimulated with LPS (100 ng/ml) for the indicated periods. Total RNA was extracted and Fra-1 mRNA expression was analyzed by quantitative reverse transcription–polymerase chain reaction using β-actin as a reference. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS. ( B ) Lysates from cells treated with LPS were isolated and immunoblotted with Fra-1 antibodies. Membrane was stripped and reprobed with β-actin antibodies. ( C ) Cells were incubated with actinomycin D (Act D), 5 µg/ml for 30 minutes and then stimulated with LPS for 3 hours; RNA was isolated, and Fra-1 mRNA expression was analyzed. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; † P ≤ 0.05, vehicle versus Act D. ( D ) The Fra-1 promoter reporter constructs bearing major cis -acting regulatory elements shown are: TPA response element (TRE); serum response element (SRE); GC-box, Sp1-binding site; and ETS-binding motif. The SRE contains the ternary complex factor binding site, the serum response factor (SRF) binding CArG site, and the activating transcription factor (ATF) or cAMP response element binding protein site. The position of these cis ). The κB element of Fra-1 bears 90% homology with a consensus κB element (5′-GGGRNWYYCC-3′). Cells were transfected with the indicated promoter reporter construct together with a reference plasmid, pRL-TK. After 24 hours, cells were cultured with medium containing 2% FBS for 3 hours and then treated with LPS for 6 hours. The luciferase activity was calculated after normalizing with Renilla values. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; † P ≤ 0.05, 92-Luc versus 132-Luc or 213-Luc. con, control; Fra-1, Fos-related antigen-1.
Figure Legend Snippet: LPS induces Fra-1 expression in alveolar macrophages. ( A ) Cells were cultured with medium containing 2% FBS for 3 hours and then stimulated with LPS (100 ng/ml) for the indicated periods. Total RNA was extracted and Fra-1 mRNA expression was analyzed by quantitative reverse transcription–polymerase chain reaction using β-actin as a reference. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS. ( B ) Lysates from cells treated with LPS were isolated and immunoblotted with Fra-1 antibodies. Membrane was stripped and reprobed with β-actin antibodies. ( C ) Cells were incubated with actinomycin D (Act D), 5 µg/ml for 30 minutes and then stimulated with LPS for 3 hours; RNA was isolated, and Fra-1 mRNA expression was analyzed. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; † P ≤ 0.05, vehicle versus Act D. ( D ) The Fra-1 promoter reporter constructs bearing major cis -acting regulatory elements shown are: TPA response element (TRE); serum response element (SRE); GC-box, Sp1-binding site; and ETS-binding motif. The SRE contains the ternary complex factor binding site, the serum response factor (SRF) binding CArG site, and the activating transcription factor (ATF) or cAMP response element binding protein site. The position of these cis ). The κB element of Fra-1 bears 90% homology with a consensus κB element (5′-GGGRNWYYCC-3′). Cells were transfected with the indicated promoter reporter construct together with a reference plasmid, pRL-TK. After 24 hours, cells were cultured with medium containing 2% FBS for 3 hours and then treated with LPS for 6 hours. The luciferase activity was calculated after normalizing with Renilla values. Data are presented as mean ± SEM ( n = 3); *P ≤ 0.05, PBS versus LPS; † P ≤ 0.05, 92-Luc versus 132-Luc or 213-Luc. con, control; Fra-1, Fos-related antigen-1.

Techniques Used: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Isolation, Incubation, Activated Clotting Time Assay, Construct, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

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    Millipore cell proliferation pbmcs
    Effect of ILT2 blockade and lenalidomide on the activation of NK cells in <t>CLL.</t> Flow cytometry analyses were conducted to evaluate NK cell expression of CD69 in <t>PBMCs</t> obtained from 14 CLL patients and stimulated with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 (10 μg/ml) and lenalidomide (LND, 1 μM) or IL-2 (50 U/ml) for 7 days. The figure shows the comparison between the surface expression of CD69 detected on NK cells (A) and leukemic cells (B) . Bars represent the MFI of CD69 ± SEM for each condition. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; ** P
    Cell Proliferation Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sterile pbs
    Multipotent adult progenitor cells <t>(MAPC)</t> administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to <t>PBS</t> and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.
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    Millipore pbs
    Phagocytes from DC-depleted mice are more effective in bacterial killing. ( A ) Splenocytes from DT-treated control (black symbols) and DC-depleted (red symbols) mice were incubated in vitro with Ye (MOI 1) for 10 min. Cells were extensively washed and either plated directly on MH agar plates or incubated further in the presence of gentamicin for the indicated time points before plating. The diagrams show the CFU as log scale (left) and the frequency of killed Ye (right) from one representative out of two experiments with quintuplicates including mean ± SD. ( B ) Viable intracellular Yersinia from sorted CD11b + Gr-1 − spleen cells or neutrophils of DT-treated control (black symbols) and DC-depleted (red symbols) mice injected with 5×10 4 Ye pYV + were analyzed by plating serial dilutions 1 and 3 dpi. CFU were analyzed per 10.000 sorted spleen cells. Data are from two independent experiments with three to six mice per group (mean ± SD). * indicates statistically significant differences (Student's t -test). ( C ) Flow cytometry analyses of ROS production in monocytes and neutrophils from DT-treated control (black symbols) and DC-depleted (red symbols) uninfected mice (left diagram) or injected for 2 h with 5×10 4 Ye pYV + (right diagram) using 2′, 7′-Dichlorofluorescin diacetate reagent (DCFD). The graph shows fold increase of median fluorescence intensity of DCFD in monocytes and neutrophils from DC-depleted mice compared to control mice. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. * indicates statistically significant differences (one-way ANOVA with Bonferroni post test). Data are from 6 independent experiments. ( D ) Splenic neutrophils from DT-treated control mice (black open circles) or DC-depleted mice (red open circles) were purified, adoptively transferred into control mice and infected with 5×10 4 Ye 30 min later. Control (black circles) or DC-depleted (red circles) mice received <t>PBS</t> instead of neutrophils. The CFU per spleen were analyzed by plating serial dilutions 1 dpi. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. Data are from one out of three independent experiments with similar results (three to five mice per group). * indicates statistically significant differences compared to control mice without adoptive transfer (one-way ANOVA with Bonferroni post test).
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    Effect of ILT2 blockade and lenalidomide on the activation of NK cells in CLL. Flow cytometry analyses were conducted to evaluate NK cell expression of CD69 in PBMCs obtained from 14 CLL patients and stimulated with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 (10 μg/ml) and lenalidomide (LND, 1 μM) or IL-2 (50 U/ml) for 7 days. The figure shows the comparison between the surface expression of CD69 detected on NK cells (A) and leukemic cells (B) . Bars represent the MFI of CD69 ± SEM for each condition. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; ** P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: Effect of ILT2 blockade and lenalidomide on the activation of NK cells in CLL. Flow cytometry analyses were conducted to evaluate NK cell expression of CD69 in PBMCs obtained from 14 CLL patients and stimulated with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 (10 μg/ml) and lenalidomide (LND, 1 μM) or IL-2 (50 U/ml) for 7 days. The figure shows the comparison between the surface expression of CD69 detected on NK cells (A) and leukemic cells (B) . Bars represent the MFI of CD69 ± SEM for each condition. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; ** P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Activation Assay, Flow Cytometry, Cytometry, Expressing, Blocking Assay

    ILT2 blockade and lenalidomide promote NK cell cytotoxicity and the elimination of primary leukemic cells from patients with CLL. PBMCs obtained from 6 CLL patients were cultured in the presence of anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) for the time points indicated. (A) The absolute numbers of leukemic cells were then analyzed after 3, 5, and 7 days of treatment by staining with anti-CD19 antibody and PKH26 microbeads as a reference. (B) The effect of ILT2 blockade and lenalidomide (7 days) on the apoptosis of leukemic cells was evaluated by annexin V assay by flow cytometry ( n = 11). (C) NK cell intracellular perforin expression was evaluated by flow cytometry analysis in PBMCs from 6 patients with CLL upon 7 days of treatment with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM). (D) NK cell cytotoxic activity against K562 leukemia cells after a week of treatment with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) was evaluated by calcein assay ( n = 5). Bars represent the mean ± SEM of the different treatments used. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: ILT2 blockade and lenalidomide promote NK cell cytotoxicity and the elimination of primary leukemic cells from patients with CLL. PBMCs obtained from 6 CLL patients were cultured in the presence of anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) for the time points indicated. (A) The absolute numbers of leukemic cells were then analyzed after 3, 5, and 7 days of treatment by staining with anti-CD19 antibody and PKH26 microbeads as a reference. (B) The effect of ILT2 blockade and lenalidomide (7 days) on the apoptosis of leukemic cells was evaluated by annexin V assay by flow cytometry ( n = 11). (C) NK cell intracellular perforin expression was evaluated by flow cytometry analysis in PBMCs from 6 patients with CLL upon 7 days of treatment with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM). (D) NK cell cytotoxic activity against K562 leukemia cells after a week of treatment with anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) was evaluated by calcein assay ( n = 5). Bars represent the mean ± SEM of the different treatments used. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Cell Culture, Blocking Assay, Staining, Annexin V Assay, Flow Cytometry, Cytometry, Expressing, Activity Assay

    Effect of lenalidomide on ILT2 expression on NK cells and B cells from healthy donors and patients with CLL. The expression of ILT2 on NK cells and B cells from healthy donors (A,B) and patients with CLL (C,D) was evaluated by flow cytometry in PBMCs obtained from 4 CLL patients and 6 controls after the treatment with different doses of lenalidomide (LND) (0.1, 1, and 10 μM) for 7 days. The figure shows the comparison of the MFI of ILT2 expression normalized to the DMSO condition ± SEM. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: Effect of lenalidomide on ILT2 expression on NK cells and B cells from healthy donors and patients with CLL. The expression of ILT2 on NK cells and B cells from healthy donors (A,B) and patients with CLL (C,D) was evaluated by flow cytometry in PBMCs obtained from 4 CLL patients and 6 controls after the treatment with different doses of lenalidomide (LND) (0.1, 1, and 10 μM) for 7 days. The figure shows the comparison of the MFI of ILT2 expression normalized to the DMSO condition ± SEM. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Expressing, Flow Cytometry, Cytometry

    Effect of lenalidomide and IL-2 on the expression of ILT2 ligands on leukemic cells. The effect of lenalidomide (LND; 1 μM) and IL-2 (50 U/mL) on the expression of classical and non-classical MHC class I molecules on leukemic cells was analyzed by flow cytometry in PBMCs obtained from 10 CLL patients. The figure shows the comparison of the expression of MHC class I (A) , HLA-E (B) , HLA-G (C) , and HLA-F (D) on leukemic cells treated with both drugs. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: Effect of lenalidomide and IL-2 on the expression of ILT2 ligands on leukemic cells. The effect of lenalidomide (LND; 1 μM) and IL-2 (50 U/mL) on the expression of classical and non-classical MHC class I molecules on leukemic cells was analyzed by flow cytometry in PBMCs obtained from 10 CLL patients. The figure shows the comparison of the expression of MHC class I (A) , HLA-E (B) , HLA-G (C) , and HLA-F (D) on leukemic cells treated with both drugs. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Expressing, Flow Cytometry, Cytometry

    ILT2 blockade and lenalidomide promote NK cell proliferation in CLL. PBMCs from 11 CLL patients were stained with CFSE and cultured in the presence of anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) for 14 days. The proliferation of NK cells and leukemic cells was analyzed by evaluating the expression of CSFE by flow cytometry. (A) Histograms show the flow cytometry profiles corresponding to CFSE expression on NK cells (CD3 − CD56 + ) and leukemic cells (CD19 + cells) of a representative CLL patient. (B,C) The histograms show the comparison of the percentage of proliferating NK cells (B) and leukemic cells (C) among the different experimental conditions analyzed. Bars represent the mean ± SEM from samples analyzed. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: ILT2 blockade and lenalidomide promote NK cell proliferation in CLL. PBMCs from 11 CLL patients were stained with CFSE and cultured in the presence of anti-ILT2 blocking antibody (10 μg/ml) or irrelevant IgG 1 and lenalidomide (LND, 1 μM) for 14 days. The proliferation of NK cells and leukemic cells was analyzed by evaluating the expression of CSFE by flow cytometry. (A) Histograms show the flow cytometry profiles corresponding to CFSE expression on NK cells (CD3 − CD56 + ) and leukemic cells (CD19 + cells) of a representative CLL patient. (B,C) The histograms show the comparison of the percentage of proliferating NK cells (B) and leukemic cells (C) among the different experimental conditions analyzed. Bars represent the mean ± SEM from samples analyzed. SEM, Standard Error of the Mean; Wilcoxon Matched-Pairs Signed Ranks test; * P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Staining, Cell Culture, Blocking Assay, Expressing, Flow Cytometry, Cytometry

    Surface ILT2 expression is increased on NK cells of CLL patients. (A) The expression of ILT2 was analyzed in PBMCs from 60 CLL patients and 25 healthy donors by flow cytometry. The histogram shows the ILT2 expression on NK cells (CD3 − CD56 + ) from a representative healthy donor and a patient with CLL. (B) The comparison between the MFI ±SEM of ILT2 surface expression on NK cells from healthy controls ( n = 25) and patients with CLL ( n = 60) is shown. (C) The comparison between the percentage of ILT2 + NK cells from healthy controls and patients is shown. Horizontal bars represent the mean ± SEM. (D) The comparison between the MFI ± SEM of ILT2 surface expression on leukemic cells and B cells from healthy controls is shown. SEM, Standard Error of the Mean; Mann-Whitney U -test; ** P

    Journal: Frontiers in Immunology

    Article Title: Ig-Like Transcript 2 (ILT2) Blockade and Lenalidomide Restore NK Cell Function in Chronic Lymphocytic Leukemia

    doi: 10.3389/fimmu.2018.02917

    Figure Lengend Snippet: Surface ILT2 expression is increased on NK cells of CLL patients. (A) The expression of ILT2 was analyzed in PBMCs from 60 CLL patients and 25 healthy donors by flow cytometry. The histogram shows the ILT2 expression on NK cells (CD3 − CD56 + ) from a representative healthy donor and a patient with CLL. (B) The comparison between the MFI ±SEM of ILT2 surface expression on NK cells from healthy controls ( n = 25) and patients with CLL ( n = 60) is shown. (C) The comparison between the percentage of ILT2 + NK cells from healthy controls and patients is shown. Horizontal bars represent the mean ± SEM. (D) The comparison between the MFI ± SEM of ILT2 surface expression on leukemic cells and B cells from healthy controls is shown. SEM, Standard Error of the Mean; Mann-Whitney U -test; ** P

    Article Snippet: Cell Proliferation PBMCs obtained from CLL patients were labeled with 1 μM 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma-Aldrich) and cultured in the presence or absence of the anti-ILT2 blocking antibody (HP-F1) and lenalidomide (1 μM) for 14 days.

    Techniques: Expressing, Flow Cytometry, Cytometry, MANN-WHITNEY

    Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to PBS and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) do not gain access to the spleen. 1 × 10 6 Qtracker ® 625-labeled MAPC were administered [intravenously (IV) or IP] to PBS and IL-7-treated mice and whole mice ( n = 3 per group) or spleen ( n = 3 per group) were harvested 48 h later. (A) CryoViz Images (left: dorsal view, right: side view. The lungs are depicted in red, liver in green, and spleen in blue). The majority of cells administered IV were detected in the lung, liver, and spleen, while MAPC administered IP were found in the peritoneal area surrounding abdominal organs. Representative images present 3D analysis of MAPC-treated mice with detected MAPC shown in yellow. (B) CryoViz Images of the spleen. MAPC IV were detected in the spleen; however, MAPC IP did not gain access to the spleen, but were detected in the omental tissue surrounding the spleen ( n = 3). 3D images show representative spleens with detected MAPC shown in yellow. (C) The fold change in the number of MAPC detected in whole mice and the total number of MAPC detected in spleens of mice at 48 h was quantified using quantification software ( n = 3). For the fold change calculation, the number of MAPC detected in the PBS + MAPC IV group was set to 1, and the fold change in the number of MAPC detected in the other groups was calculated based on this.

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: Labeling, Mouse Assay, Software

    Multipotent adult progenitor cells (MAPC) suppress IL-7-induced interferon-γ (IFN-γ) production by T cells in vivo . Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (A) The frequency of IFN-γ producing CD4 + and CD8 + in the spleen was significantly increased following IL-7 administration. Both MAPC IP and MAPC IV reduced IFN-γ production by T cells (PBS: n = 8, PBS + MAPC IP: n = 12, PBS + MAPC IV: n = 8, IL-7: n = 6, IL-7 + MAPC IP: n = 11, IL-7 + MAPC IV: n = 8). (B) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of IFN-γ production by CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 8, PBS + MAPC IP: n = 11, PBS + MAPC IV: n = 7, IL-7: n = 7, IL-7 + MAPC IP: n = 10, and IL-7 + MAPC IV: n = 7). Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) suppress IL-7-induced interferon-γ (IFN-γ) production by T cells in vivo . Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (A) The frequency of IFN-γ producing CD4 + and CD8 + in the spleen was significantly increased following IL-7 administration. Both MAPC IP and MAPC IV reduced IFN-γ production by T cells (PBS: n = 8, PBS + MAPC IP: n = 12, PBS + MAPC IV: n = 8, IL-7: n = 6, IL-7 + MAPC IP: n = 11, IL-7 + MAPC IV: n = 8). (B) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of IFN-γ production by CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 8, PBS + MAPC IP: n = 11, PBS + MAPC IV: n = 7, IL-7: n = 7, IL-7 + MAPC IP: n = 10, and IL-7 + MAPC IV: n = 7). Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: In Vivo, Recombinant

    Multipotent adult progenitor cells (MAPC) have no effect on T cell proliferation following lymphodepletion. 50 mg/kg anti-thymocyte globulin (ATG) was administered intraperitoneally (IP) on days 0 and 3. (A) Spleens and lymph nodes were harvested on days 4 and 7, and total numbers of CD4 + and CD8 + cells were quantified using counting beads and flow cytometry. (B) Schematic timeline of experiments with MAPC (1 × 10 6 ) administration [IP or intravenously (IV)] to the ATG model on day 4. (C) MAPC administered IV or IP had no effect on the proliferation of either CD4 + or CD8 + T cells in the spleens and lymph nodes following ATG administration (spleens; PBS: n = 10, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 8 and lymph nodes; PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 9, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. *

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) have no effect on T cell proliferation following lymphodepletion. 50 mg/kg anti-thymocyte globulin (ATG) was administered intraperitoneally (IP) on days 0 and 3. (A) Spleens and lymph nodes were harvested on days 4 and 7, and total numbers of CD4 + and CD8 + cells were quantified using counting beads and flow cytometry. (B) Schematic timeline of experiments with MAPC (1 × 10 6 ) administration [IP or intravenously (IV)] to the ATG model on day 4. (C) MAPC administered IV or IP had no effect on the proliferation of either CD4 + or CD8 + T cells in the spleens and lymph nodes following ATG administration (spleens; PBS: n = 10, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 8 and lymph nodes; PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 9, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. *

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: Flow Cytometry, Cytometry

    Multipotent adult progenitor cells (MAPC) promote Treg frequency and suppress interferon-γ (IFN-γ) production by T cells following lymphodepletion. (A) Bar graph and representative FACS plots demonstrate that anti-thymocyte globulin (ATG) increased the frequency of CD4 + , CD25 + , and FoxP3 + T cells in the spleen and lymph nodes, and MAPC cells administered intraperitoneally (MAPC IP) further increased this in the lymph nodes [PBS: n = 13, ATG: n = 13, ATG + MAPC IP: n = 11, and ATG + MAPC cells administered intravenously (MAPC IV): n = 13]. (B) MAPC IP reduced the frequency of IFN-γ producing CD4 + and CD8 + T cells in the spleen (PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 10). (C) MAPC IP reduced the frequency of IFN-γ producing CD8 + T cells but not CD4 + T cells in the lymph nodes (PBS: n = 12, ATG: n = 10, ATG + MAPC IP: n = 11, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) promote Treg frequency and suppress interferon-γ (IFN-γ) production by T cells following lymphodepletion. (A) Bar graph and representative FACS plots demonstrate that anti-thymocyte globulin (ATG) increased the frequency of CD4 + , CD25 + , and FoxP3 + T cells in the spleen and lymph nodes, and MAPC cells administered intraperitoneally (MAPC IP) further increased this in the lymph nodes [PBS: n = 13, ATG: n = 13, ATG + MAPC IP: n = 11, and ATG + MAPC cells administered intravenously (MAPC IV): n = 13]. (B) MAPC IP reduced the frequency of IFN-γ producing CD4 + and CD8 + T cells in the spleen (PBS: n = 8, ATG: n = 10, ATG + MAPC IP: n = 10, and ATG + MAPC IV: n = 10). (C) MAPC IP reduced the frequency of IFN-γ producing CD8 + T cells but not CD4 + T cells in the lymph nodes (PBS: n = 12, ATG: n = 10, ATG + MAPC IP: n = 11, and ATG + MAPC IV: n = 10). Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: FACS

    Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) require prostaglandin E2 (PGE2) to suppress interferon-γ (IFN-γ) production by T cells. 100 mg/kg anti-thymocyte globulin (ATG) or control serum was administered over two doses given on days 0 and 3, followed by the administration of MAPC IP on day 4, and indomethacin (Indo) on days 4, 5, and 6. Spleens and lymph nodes were harvested on day 7 and examined for the production of IFN-γ by T cells. Bar graphs show that suppression of IFN-γ production by CD4 + and CD8 + T cells in the (A) spleen [PBS: n = 7, ATG: n = 7, ATG + MAPC IP: n = 9, and ATG + MAPC intravenous (IV): n = 7] and (B) lymph nodes (PBS: n = 4, ATG: n = 5, ATG + MAPC IP: n = 5, and ATG + MAPC IV: n = 4) was inhibited when Indo was administered on days 4, 5, and 6 of the lymphodepletion model. Results are indicative of two independent experiments using two MAPC donors. * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) administered intraperitoneally (IP) require prostaglandin E2 (PGE2) to suppress interferon-γ (IFN-γ) production by T cells. 100 mg/kg anti-thymocyte globulin (ATG) or control serum was administered over two doses given on days 0 and 3, followed by the administration of MAPC IP on day 4, and indomethacin (Indo) on days 4, 5, and 6. Spleens and lymph nodes were harvested on day 7 and examined for the production of IFN-γ by T cells. Bar graphs show that suppression of IFN-γ production by CD4 + and CD8 + T cells in the (A) spleen [PBS: n = 7, ATG: n = 7, ATG + MAPC IP: n = 9, and ATG + MAPC intravenous (IV): n = 7] and (B) lymph nodes (PBS: n = 4, ATG: n = 5, ATG + MAPC IP: n = 5, and ATG + MAPC IV: n = 4) was inhibited when Indo was administered on days 4, 5, and 6 of the lymphodepletion model. Results are indicative of two independent experiments using two MAPC donors. * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques:

    Multipotent adult progenitor cells (MAPC) suppress IL-7-driven proliferation of T cells in vivo . (A) Schematic timeline of IL-7-driven homeostatic proliferation experiments. Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC cells were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (B) Bar graphs demonstrating that both MAPC IV and MAPC IP reduce the frequency of Ki67 + CD4 + and CD8 + cells in the spleen (PBS: n = 13, PBS + MAPC IP: n = 17, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13) (C) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of Ki67 + CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 12, PBS + MAPC IP: n = 15, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13). Results are indicative of three independent experiments using three MAPC donors * p

    Journal: Frontiers in Immunology

    Article Title: Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner

    doi: 10.3389/fimmu.2018.00645

    Figure Lengend Snippet: Multipotent adult progenitor cells (MAPC) suppress IL-7-driven proliferation of T cells in vivo . (A) Schematic timeline of IL-7-driven homeostatic proliferation experiments. Recombinant IL-7 conjugated to M25 or PBS was administered intraperitoneally (IP) on days 0, 2, and 4. 1 × 10 6 MAPC cells were administered IP or intravenously (IV) on day 1. Lymph nodes and spleens were harvested on day 5. (B) Bar graphs demonstrating that both MAPC IV and MAPC IP reduce the frequency of Ki67 + CD4 + and CD8 + cells in the spleen (PBS: n = 13, PBS + MAPC IP: n = 17, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13) (C) Bar graphs demonstrating that MAPC IP but not MAPC IV reduce the frequency of Ki67 + CD4 + and CD8 + T cells in the lymph nodes (PBS: n = 12, PBS + MAPC IP: n = 15, PBS + MAPC IV: n = 13, IL-7: n = 12, IL-7 + MAPC IP: n = 17, and IL-7 + MAPC IV: n = 13). Results are indicative of three independent experiments using three MAPC donors * p

    Article Snippet: MAPC cells were then counted and washed twice in sterile PBS (Sigma-Aldrich).

    Techniques: In Vivo, Recombinant

    Effect of allergen sensitization on phylogenetic diversity. A , Relative abundance of the different microbial phyla in fecal samples of WT and Il4raF709 mice that were either sham sensitized with PBS or sensitized with OVA or OVA/SEB. The combined profile

    Journal: The Journal of allergy and clinical immunology

    Article Title: A microbiota signature associated with experimental food allergy promotes allergic sensitization and anaphylaxis

    doi: 10.1016/j.jaci.2012.10.026

    Figure Lengend Snippet: Effect of allergen sensitization on phylogenetic diversity. A , Relative abundance of the different microbial phyla in fecal samples of WT and Il4raF709 mice that were either sham sensitized with PBS or sensitized with OVA or OVA/SEB. The combined profile

    Article Snippet: For sensitization, female WT and Il4raF709 mice were treated intragastrically with sterile PBS or 100 μg of OVA alone or together with 10 μg of staphylococcal enterotoxin B (SEB; Sigma-Aldrich, St Louis, Mo) in 100 μL of sterile PBS (saline) once weekly for 8 weeks.

    Techniques: Mouse Assay

    Oral sensitization and anaphylaxis in Il4raF709 mice: prevention of oral sensitization by antigen-specific Treg cells. A , Il4raF709 and WT BALB/c control mice were either sham sensitized with PBS or sensitized with OVA (100 μg) or OVA/SEB (100

    Journal: The Journal of allergy and clinical immunology

    Article Title: A microbiota signature associated with experimental food allergy promotes allergic sensitization and anaphylaxis

    doi: 10.1016/j.jaci.2012.10.026

    Figure Lengend Snippet: Oral sensitization and anaphylaxis in Il4raF709 mice: prevention of oral sensitization by antigen-specific Treg cells. A , Il4raF709 and WT BALB/c control mice were either sham sensitized with PBS or sensitized with OVA (100 μg) or OVA/SEB (100

    Article Snippet: For sensitization, female WT and Il4raF709 mice were treated intragastrically with sterile PBS or 100 μg of OVA alone or together with 10 μg of staphylococcal enterotoxin B (SEB; Sigma-Aldrich, St Louis, Mo) in 100 μL of sterile PBS (saline) once weekly for 8 weeks.

    Techniques: Mouse Assay

    Phagocytes from DC-depleted mice are more effective in bacterial killing. ( A ) Splenocytes from DT-treated control (black symbols) and DC-depleted (red symbols) mice were incubated in vitro with Ye (MOI 1) for 10 min. Cells were extensively washed and either plated directly on MH agar plates or incubated further in the presence of gentamicin for the indicated time points before plating. The diagrams show the CFU as log scale (left) and the frequency of killed Ye (right) from one representative out of two experiments with quintuplicates including mean ± SD. ( B ) Viable intracellular Yersinia from sorted CD11b + Gr-1 − spleen cells or neutrophils of DT-treated control (black symbols) and DC-depleted (red symbols) mice injected with 5×10 4 Ye pYV + were analyzed by plating serial dilutions 1 and 3 dpi. CFU were analyzed per 10.000 sorted spleen cells. Data are from two independent experiments with three to six mice per group (mean ± SD). * indicates statistically significant differences (Student's t -test). ( C ) Flow cytometry analyses of ROS production in monocytes and neutrophils from DT-treated control (black symbols) and DC-depleted (red symbols) uninfected mice (left diagram) or injected for 2 h with 5×10 4 Ye pYV + (right diagram) using 2′, 7′-Dichlorofluorescin diacetate reagent (DCFD). The graph shows fold increase of median fluorescence intensity of DCFD in monocytes and neutrophils from DC-depleted mice compared to control mice. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. * indicates statistically significant differences (one-way ANOVA with Bonferroni post test). Data are from 6 independent experiments. ( D ) Splenic neutrophils from DT-treated control mice (black open circles) or DC-depleted mice (red open circles) were purified, adoptively transferred into control mice and infected with 5×10 4 Ye 30 min later. Control (black circles) or DC-depleted (red circles) mice received PBS instead of neutrophils. The CFU per spleen were analyzed by plating serial dilutions 1 dpi. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. Data are from one out of three independent experiments with similar results (three to five mice per group). * indicates statistically significant differences compared to control mice without adoptive transfer (one-way ANOVA with Bonferroni post test).

    Journal: PLoS Pathogens

    Article Title: Depletion of Dendritic Cells Enhances Innate Anti-Bacterial Host Defense through Modulation of Phagocyte Homeostasis

    doi: 10.1371/journal.ppat.1002552

    Figure Lengend Snippet: Phagocytes from DC-depleted mice are more effective in bacterial killing. ( A ) Splenocytes from DT-treated control (black symbols) and DC-depleted (red symbols) mice were incubated in vitro with Ye (MOI 1) for 10 min. Cells were extensively washed and either plated directly on MH agar plates or incubated further in the presence of gentamicin for the indicated time points before plating. The diagrams show the CFU as log scale (left) and the frequency of killed Ye (right) from one representative out of two experiments with quintuplicates including mean ± SD. ( B ) Viable intracellular Yersinia from sorted CD11b + Gr-1 − spleen cells or neutrophils of DT-treated control (black symbols) and DC-depleted (red symbols) mice injected with 5×10 4 Ye pYV + were analyzed by plating serial dilutions 1 and 3 dpi. CFU were analyzed per 10.000 sorted spleen cells. Data are from two independent experiments with three to six mice per group (mean ± SD). * indicates statistically significant differences (Student's t -test). ( C ) Flow cytometry analyses of ROS production in monocytes and neutrophils from DT-treated control (black symbols) and DC-depleted (red symbols) uninfected mice (left diagram) or injected for 2 h with 5×10 4 Ye pYV + (right diagram) using 2′, 7′-Dichlorofluorescin diacetate reagent (DCFD). The graph shows fold increase of median fluorescence intensity of DCFD in monocytes and neutrophils from DC-depleted mice compared to control mice. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. * indicates statistically significant differences (one-way ANOVA with Bonferroni post test). Data are from 6 independent experiments. ( D ) Splenic neutrophils from DT-treated control mice (black open circles) or DC-depleted mice (red open circles) were purified, adoptively transferred into control mice and infected with 5×10 4 Ye 30 min later. Control (black circles) or DC-depleted (red circles) mice received PBS instead of neutrophils. The CFU per spleen were analyzed by plating serial dilutions 1 dpi. Each symbol represents an individual mouse; horizontal lines indicate the mean ± SD. Data are from one out of three independent experiments with similar results (three to five mice per group). * indicates statistically significant differences compared to control mice without adoptive transfer (one-way ANOVA with Bonferroni post test).

    Article Snippet: After blocking with Fc-block (hybridoma supernatant from 2.4G2 cell line producing anti-FcγRII/III mAb) in PBS containing 10% fetal bovine serum and 5% normal goat serum (Sigma-Aldrich) slides were incubated with polyclonal rabbit anti-yersinia antibodies (WA-v; 5 µg/ml) in PBS containing 10% fetal bovine serum and 5% normal goat serum for 30 min at room temperature.

    Techniques: Mouse Assay, Incubation, In Vitro, Injection, Flow Cytometry, Cytometry, Fluorescence, Purification, Infection, Adoptive Transfer Assay