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Pharmingen monensin
Effect of absence of CD28 on the development of OVA-induced eosinophilic airway inflammation. On day 0, CD28 +/+ and CD28 –/– mice received 5 × 10 5 OVA-DC from wild-type mice. Mice were subsequently exposed to OVA aerosol from day 14 to day 20 and BALF was recovered 24 hours after the last aerosol challenge. ( a ) Differential cell counting on BALF. Results represent means ± SEM from six mice per group. ( b ) Intracellular staining for cytokines on BALF T cells. Cells were restimulated with PMA/ionomycin in the presence of <t>monensin</t> and stained for IFN-γ and IL-4. Percentage expression of each cytokine on CD3 + CD4 + cells is indicated.
Monensin, supplied by Pharmingen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation"

Article Title: Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation

Journal: Journal of Clinical Investigation

doi:

Effect of absence of CD28 on the development of OVA-induced eosinophilic airway inflammation. On day 0, CD28 +/+ and CD28 –/– mice received 5 × 10 5 OVA-DC from wild-type mice. Mice were subsequently exposed to OVA aerosol from day 14 to day 20 and BALF was recovered 24 hours after the last aerosol challenge. ( a ) Differential cell counting on BALF. Results represent means ± SEM from six mice per group. ( b ) Intracellular staining for cytokines on BALF T cells. Cells were restimulated with PMA/ionomycin in the presence of monensin and stained for IFN-γ and IL-4. Percentage expression of each cytokine on CD3 + CD4 + cells is indicated.
Figure Legend Snippet: Effect of absence of CD28 on the development of OVA-induced eosinophilic airway inflammation. On day 0, CD28 +/+ and CD28 –/– mice received 5 × 10 5 OVA-DC from wild-type mice. Mice were subsequently exposed to OVA aerosol from day 14 to day 20 and BALF was recovered 24 hours after the last aerosol challenge. ( a ) Differential cell counting on BALF. Results represent means ± SEM from six mice per group. ( b ) Intracellular staining for cytokines on BALF T cells. Cells were restimulated with PMA/ionomycin in the presence of monensin and stained for IFN-γ and IL-4. Percentage expression of each cytokine on CD3 + CD4 + cells is indicated.

Techniques Used: Mouse Assay, Cell Counting, Staining, Expressing

2) Product Images from "In Vivo Function of an Interleukin 2 Receptor ? Chain (IL-2R?)/IL-4R? Cytokine Receptor Chimera Potentiates Allergic Airway Disease "

Article Title: In Vivo Function of an Interleukin 2 Receptor ? Chain (IL-2R?)/IL-4R? Cytokine Receptor Chimera Potentiates Allergic Airway Disease

Journal: The Journal of Experimental Medicine

doi:

Enhanced Th2 cytokines in early primary responses. ( A ) Lymph node cells, as in Fig. 5 , were stimulated 48 h as indicated and supernatants were collected. IL-4 and IFN-γ levels in supernatants were measured by ELISA. These figures are representative of three independent experiments; additional experiments using preparations of lymph node cells from individual mice of each subline showed no differences in the penetrance of the observed effects on cytokine (IL-4, IFN-γ) production. ( B ) Lymph node cells, as in Fig. 5 , were stimulated with immobilized anti-CD3 (10 μg/ml), anti-CD28 (2 μg/ml), and recombinant mouse IL-2 (20 ng/ml) for 40 h. Monensin was added to the cell culture in the last 4 h. Cells were stained with anti-CD4-biotin and streptavidin-PerCP, permeabilized, and stained with both anti-IL-4–PE and anti-IFN-γ-FITC, followed by FACS ® analysis. These figures are representative of histograms in two independent experiments.
Figure Legend Snippet: Enhanced Th2 cytokines in early primary responses. ( A ) Lymph node cells, as in Fig. 5 , were stimulated 48 h as indicated and supernatants were collected. IL-4 and IFN-γ levels in supernatants were measured by ELISA. These figures are representative of three independent experiments; additional experiments using preparations of lymph node cells from individual mice of each subline showed no differences in the penetrance of the observed effects on cytokine (IL-4, IFN-γ) production. ( B ) Lymph node cells, as in Fig. 5 , were stimulated with immobilized anti-CD3 (10 μg/ml), anti-CD28 (2 μg/ml), and recombinant mouse IL-2 (20 ng/ml) for 40 h. Monensin was added to the cell culture in the last 4 h. Cells were stained with anti-CD4-biotin and streptavidin-PerCP, permeabilized, and stained with both anti-IL-4–PE and anti-IFN-γ-FITC, followed by FACS ® analysis. These figures are representative of histograms in two independent experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay, Mouse Assay, Recombinant, Cell Culture, Staining, FACS

3) Product Images from "Natural killer cell function is altered during the primary response of aged mice to influenza infection"

Article Title: Natural killer cell function is altered during the primary response of aged mice to influenza infection

Journal: Mechanisms of ageing and development

doi: 10.1016/j.mad.2011.08.005

Aged NK cells have impaired function against YAC-1 cells after influenza infection Single cell suspensions from lungs and spleens of uninfected and infected young and aged mice were incubated with YAC-1 cells at an effector to target ratio of 10:1 in the presence of brefeldin, monensin and FITC-CD107a antibody for 4 hours. Bars represent means ± SEM of CD107a+ and IFN-γ+ NK cells; data shown are from one representative of two performed experiments, n=5 per group per experiment. Asterisks, (*), indicate statistical differences between young and aged mice at that time point (t-test, p
Figure Legend Snippet: Aged NK cells have impaired function against YAC-1 cells after influenza infection Single cell suspensions from lungs and spleens of uninfected and infected young and aged mice were incubated with YAC-1 cells at an effector to target ratio of 10:1 in the presence of brefeldin, monensin and FITC-CD107a antibody for 4 hours. Bars represent means ± SEM of CD107a+ and IFN-γ+ NK cells; data shown are from one representative of two performed experiments, n=5 per group per experiment. Asterisks, (*), indicate statistical differences between young and aged mice at that time point (t-test, p

Techniques Used: Infection, Mouse Assay, Incubation, T-Test

4) Product Images from "Antigen-specific dose-dependent system for the study of an inheritable and reversible phenotype in mouse CD4+ T cells"

Article Title: Antigen-specific dose-dependent system for the study of an inheritable and reversible phenotype in mouse CD4+ T cells

Journal: Immunology

doi: 10.1046/j.1365-2567.2002.01540.x

Reduced levels of intracellular IL-2 in high dose peptide stimulated TEa cells. At days 2 and 3 of a secondary challenge, after 7-hr incubation with the inhibitor monensin, viable cells were collected by Ficoll centrifugation and the accumulated intracellular expression of IL-2 was measured by FACS analysis. To establish the background level of intracellular IL-2 accumulation in nonproliferating cells, an equivalent amount of control cells were incubated in the absence of antigenic peptide (non-stimulated cells). Dot-plots show TEa cells (Vα-2-FITC fluorescence, x -axis, arbitrary units) versus IL-2 producing cells (IL-2-PE-Cy5 fluorescence, y -axis, arbitrary units). Panels are a representative experiment from two separate experiments.
Figure Legend Snippet: Reduced levels of intracellular IL-2 in high dose peptide stimulated TEa cells. At days 2 and 3 of a secondary challenge, after 7-hr incubation with the inhibitor monensin, viable cells were collected by Ficoll centrifugation and the accumulated intracellular expression of IL-2 was measured by FACS analysis. To establish the background level of intracellular IL-2 accumulation in nonproliferating cells, an equivalent amount of control cells were incubated in the absence of antigenic peptide (non-stimulated cells). Dot-plots show TEa cells (Vα-2-FITC fluorescence, x -axis, arbitrary units) versus IL-2 producing cells (IL-2-PE-Cy5 fluorescence, y -axis, arbitrary units). Panels are a representative experiment from two separate experiments.

Techniques Used: Incubation, Centrifugation, Expressing, FACS, Fluorescence

5) Product Images from "Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo"

Article Title: Interleukin-4 Diminishes CD8+ Respiratory Syncytial Virus-Specific Cytotoxic T-Lymphocyte Activity In Vivo

Journal: Journal of Virology

doi:

Intracellular cytokine staining of M2 peptide-stimulated spleen cells. Mice were primarily infected (1° CTL) or immunized (2° CTL) with vvM2 or vvM2/IL-4; 2 × 10 6 spleen cells were stimulated with FLU 147-155 (TYQRTRALV) or RSV M2 82-90 (SYIGSINNI) peptide for 8 h in the presence of monensin and analyzed for IFN-γ production by flow cytometry. Data for primary CTL are representative of averages compiled from five independent experiments with n = 4 or 5 per group; data for secondary CTL are representative of two independent experiments, n = 4 ( P > 0.05 between groups for both primary and secondary CTL responses).
Figure Legend Snippet: Intracellular cytokine staining of M2 peptide-stimulated spleen cells. Mice were primarily infected (1° CTL) or immunized (2° CTL) with vvM2 or vvM2/IL-4; 2 × 10 6 spleen cells were stimulated with FLU 147-155 (TYQRTRALV) or RSV M2 82-90 (SYIGSINNI) peptide for 8 h in the presence of monensin and analyzed for IFN-γ production by flow cytometry. Data for primary CTL are representative of averages compiled from five independent experiments with n = 4 or 5 per group; data for secondary CTL are representative of two independent experiments, n = 4 ( P > 0.05 between groups for both primary and secondary CTL responses).

Techniques Used: Staining, Mouse Assay, Infection, CTL Assay, Flow Cytometry, Cytometry

6) Product Images from "Production of Tumor Necrosis Factor Alpha in Human T Lymphocytes by Staphylococcal Enterotoxin B Correlates with Toxin-Induced Proliferation and Is Regulated through Protein Kinase C"

Article Title: Production of Tumor Necrosis Factor Alpha in Human T Lymphocytes by Staphylococcal Enterotoxin B Correlates with Toxin-Induced Proliferation and Is Regulated through Protein Kinase C

Journal: Infection and Immunity

doi:

FACScan analysis of TNF-α expression in monocytes and lymphocytes. Monocytes and lymphocytes (5 × 10 5 each) were mixed and incubated for 6 h with 100 ng of SEB per ml in the presence of 2 μM monensin. Samples were fixed and then permeabilized and stained with cell-type-specific antibody and analyzed on a FACScan flow cytometer. Negative controls (A and C) were without SEB. Samples were stained with anti-CD3 or anti-CD14 antisera to identify the following: lymphocytes (A), lymphocytes plus SEB (B), monocytes (C), and monocytes plus SEB (D).
Figure Legend Snippet: FACScan analysis of TNF-α expression in monocytes and lymphocytes. Monocytes and lymphocytes (5 × 10 5 each) were mixed and incubated for 6 h with 100 ng of SEB per ml in the presence of 2 μM monensin. Samples were fixed and then permeabilized and stained with cell-type-specific antibody and analyzed on a FACScan flow cytometer. Negative controls (A and C) were without SEB. Samples were stained with anti-CD3 or anti-CD14 antisera to identify the following: lymphocytes (A), lymphocytes plus SEB (B), monocytes (C), and monocytes plus SEB (D).

Techniques Used: Expressing, Incubation, Staining, Flow Cytometry, Cytometry

7) Product Images from "Expression of Interleukin 9 in the Lungs of Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness "

Article Title: Expression of Interleukin 9 in the Lungs of Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness

Journal: The Journal of Experimental Medicine

doi:

Intracellular detection of IL-4 and INF-γ in lymphocytes from lung lavage fluid of IL-9–expressing mice. Data shown are generated by three-color flow cytometric analysis of total lung lavage cells and pooled from four IL-9–expressing littermates after short-term culture in the presence of PMA, ionomycin, and monensin. Dot-blots were gated on CD4 + lymphocytes ( A ) or CD8 + lymphocytes ( B ). Number of cells staining for each cytokine are expressed as a percentage of CD4 + or CD8 + cells.
Figure Legend Snippet: Intracellular detection of IL-4 and INF-γ in lymphocytes from lung lavage fluid of IL-9–expressing mice. Data shown are generated by three-color flow cytometric analysis of total lung lavage cells and pooled from four IL-9–expressing littermates after short-term culture in the presence of PMA, ionomycin, and monensin. Dot-blots were gated on CD4 + lymphocytes ( A ) or CD8 + lymphocytes ( B ). Number of cells staining for each cytokine are expressed as a percentage of CD4 + or CD8 + cells.

Techniques Used: Expressing, Mouse Assay, Generated, Flow Cytometry, Staining

8) Product Images from "Lack of CD4+ T Cells Does Not Affect Induction of CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection"

Article Title: Lack of CD4+ T Cells Does Not Affect Induction of CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

Journal: Infection and Immunity

doi:

Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10 7 spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4 −/− (D), CD8 −/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for CD4 + (A and B) or CD8 + (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P
Figure Legend Snippet: Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10 7 spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4 −/− (D), CD8 −/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for CD4 + (A and B) or CD8 + (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P

Techniques Used: Staining, Knock-Out, Mouse Assay, Infection, Cell Culture, In Vitro, Labeling

9) Product Images from "Interleukin-8 and Growth-Regulated Oncogene Alpha Mediate Angiogenesis in Kaposi's Sarcoma"

Article Title: Interleukin-8 and Growth-Regulated Oncogene Alpha Mediate Angiogenesis in Kaposi's Sarcoma

Journal: Journal of Virology

doi: 10.1128/JVI.76.22.11570-11583.2002

Intracellular IL-8 is present in the monocytic subpopulation of PBMC exposed to HIV-1. PBMC were treated with monensin (control) along with HIV-1 BaL , HIV-1 BRU , or TNF-α (100 ng/ml). PBMC were harvested after 6 h and analyzed for intracellular IL-8 protein by flow cytometry. (A) Lymphocyte and monocyte subpopulations were gated according to the pattern of forward scatter and side scatter. (B) Background staining in the lymphocytes and monocytes was determined by incubation with phycoerythrin-mouse IgG1 isotype control (mouse IgG-PE) and FITC-mouse IgG2a isotype control (mouse IgG-FITC). The histograms show fluorescence intensity on logarithmic scales along the x axis (FITC) and y axis (phycoerythrin). The percentage of FITC-positive cells is indicated for both the double-positive and single-positive quadrants. (C) Lymphocytes and monocytes were stained with an FITC-conjugated mouse anti-human IL-8 antibody (IL-8-FITC) and either phycoerythrin-conjugated mouse anti-human CD4 (CD4-PE) or phycoerythrin-conjugated mouse anti-human CD14 (CD14-PE), respectively. The percentage of CD4 + and CD14 + cells staining positive for IL-8 in each condition is indicated. Data shown are representative of four independent experiments.
Figure Legend Snippet: Intracellular IL-8 is present in the monocytic subpopulation of PBMC exposed to HIV-1. PBMC were treated with monensin (control) along with HIV-1 BaL , HIV-1 BRU , or TNF-α (100 ng/ml). PBMC were harvested after 6 h and analyzed for intracellular IL-8 protein by flow cytometry. (A) Lymphocyte and monocyte subpopulations were gated according to the pattern of forward scatter and side scatter. (B) Background staining in the lymphocytes and monocytes was determined by incubation with phycoerythrin-mouse IgG1 isotype control (mouse IgG-PE) and FITC-mouse IgG2a isotype control (mouse IgG-FITC). The histograms show fluorescence intensity on logarithmic scales along the x axis (FITC) and y axis (phycoerythrin). The percentage of FITC-positive cells is indicated for both the double-positive and single-positive quadrants. (C) Lymphocytes and monocytes were stained with an FITC-conjugated mouse anti-human IL-8 antibody (IL-8-FITC) and either phycoerythrin-conjugated mouse anti-human CD4 (CD4-PE) or phycoerythrin-conjugated mouse anti-human CD14 (CD14-PE), respectively. The percentage of CD4 + and CD14 + cells staining positive for IL-8 in each condition is indicated. Data shown are representative of four independent experiments.

Techniques Used: Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

Related Articles

Concentration Assay:

Article Title: T lymphocytes from granulocyte colony-stimulating factor–/– mice produce large quantities of interferon-? in a chronic infection model
Article Snippet: ICC staining was used to determine the frequency of IFN-γ-producing CD4+ and CD8+ T cells according to previous methods. .. Spleen cells were transferred to 24-well culture plates (Costar, Corning Inc., Corning, NY) at a concentration of 2 × 106 cells in 1 ml, the T cells stimulated with protein G purified immobilized anti-CD3 mAb (2·5 µg/ml) in the presence of 2 µ m monensin (PharMingen) for 6 hr at 37° in 5% CO2 . .. Aliquots of 1 × 106 cells were transferred to FACS tubes (Falcon 12 × 75 mm round-bottom tubes, Becton Dickinson), and washed with staining buffer (PBS, 1% heat-inactivated FCS, 0·1% (w/v) sodium azide).

Purification:

Article Title: T lymphocytes from granulocyte colony-stimulating factor–/– mice produce large quantities of interferon-? in a chronic infection model
Article Snippet: ICC staining was used to determine the frequency of IFN-γ-producing CD4+ and CD8+ T cells according to previous methods. .. Spleen cells were transferred to 24-well culture plates (Costar, Corning Inc., Corning, NY) at a concentration of 2 × 106 cells in 1 ml, the T cells stimulated with protein G purified immobilized anti-CD3 mAb (2·5 µg/ml) in the presence of 2 µ m monensin (PharMingen) for 6 hr at 37° in 5% CO2 . .. Aliquots of 1 × 106 cells were transferred to FACS tubes (Falcon 12 × 75 mm round-bottom tubes, Becton Dickinson), and washed with staining buffer (PBS, 1% heat-inactivated FCS, 0·1% (w/v) sodium azide).

Article Title: Antigen-specific dose-dependent system for the study of an inheritable and reversible phenotype in mouse CD4+ T cells
Article Snippet: Mouse CD4+ T-cell subset enrichment columns and recombinant murine IL-2 were purchased from R & D Systems (Minneapolis, MN). .. Anti-mouse Vα-2-fluorescein isothiocyanate (Vα-2-FITC) Clone B20·1, anti-bromodeoxyuridine-FITC (BrdU-FITC) clone 3D4, anti-mouse CD4-phycoerythrin (PE), CD3ε-PE, biotinylated anti-mouse IL-2 clone JES6-544, biotinylated anti-mouse immunoglobulin G2b (IgG2b), purified anti-mouse CD16/CD32 (FcγIII/II receptor), and anti-mouse p27Kip1 monoclonal antibodies (mAbs), monensin, streptavidin–PE, streptavidin–Cy–chrome (CY-5) conjugates, and annexin V–FITC were all purchased from Pharmingen (San Diego, CA). .. Affinity purified polyclonal antibodies against anticyclin E were generated as previously described.

Staining:

Article Title: Generalized Immunological Decline during Long-Term Experimental Infection with Mycobacterium avium
Article Snippet: Lungs were removed from groups of three mice at different stages of infection and subjected to enzymatic digestion to generate single-cell suspensions ( ). .. Cells were stimulated with immobilized anti-CD3 monoclonal antibody (MAb) (2.5 μg of protein G-purified antibody 145-2C11 per ml) in the presence of 2 μM monensin (Pharmingen) for 6 h, and then stained with either phycoerythrin (PE)-conjugated anti-CD4 (H129.19) or anti-CD8 (53-6.7) MAb, fixed and permeabilized, and stained with fluorescein isothiocyanate (FITC)-conjugated anti-IFN-γ MAb (XMG1.2). .. Because the majority of lung cells were Fc receptor-bearing macrophages, high levels of nonspecific staining made it difficult to analyze whole populations of lung cells by flow cytometry.

Article Title: Interleukin-10 Secretion Differentiates Dendritic Cells from Human Liver and Skin
Article Snippet: Cells were incubated in 10% mouse serum before final incubation in phycoerythrin-class II antibody. .. For intracellular cytokine staining DCs were stimulated for 5 hours with 5 ng/ml of PMA and 500 ng/ml of ionomycin (Sigma), and cytokine export blocked with monensin (Pharmingen) used according to the manufacturer’s instructions. .. Cell surface markers were stained, eg, CD11c followed by rabbit anti-mouse fluorescein isothiocyanate, before fixation and permeabilization, and phycoerythrin-conjugated antibodies to cytokine or the recommended control antibodies from Pharmingen were used to stain permeabilized cells.

Cell Culture:

Article Title: Induction of Cell-Mediated Immunity to Staphylococcus aureus in the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant
Article Snippet: Cytokine production by different lymphocyte subpopulations was evaluated at the single-cell level by intracellular staining by using the Citofix/Cytoperm Plus kit (Pharmingen) according to the manufacturer's instructions. .. Briefly, cultured cells or freshly isolated cells from mice challenged with wt-S. aureus were incubated consecutively with monensin, Cytofix/Cytoperm solution (Pharmingen), and FITC-conjugated anti-IFN-γ (Pharmingen). ..

Isolation:

Article Title: Induction of Cell-Mediated Immunity to Staphylococcus aureus in the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant
Article Snippet: Cytokine production by different lymphocyte subpopulations was evaluated at the single-cell level by intracellular staining by using the Citofix/Cytoperm Plus kit (Pharmingen) according to the manufacturer's instructions. .. Briefly, cultured cells or freshly isolated cells from mice challenged with wt-S. aureus were incubated consecutively with monensin, Cytofix/Cytoperm solution (Pharmingen), and FITC-conjugated anti-IFN-γ (Pharmingen). ..

Mouse Assay:

Article Title: Induction of Cell-Mediated Immunity to Staphylococcus aureus in the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant
Article Snippet: Cytokine production by different lymphocyte subpopulations was evaluated at the single-cell level by intracellular staining by using the Citofix/Cytoperm Plus kit (Pharmingen) according to the manufacturer's instructions. .. Briefly, cultured cells or freshly isolated cells from mice challenged with wt-S. aureus were incubated consecutively with monensin, Cytofix/Cytoperm solution (Pharmingen), and FITC-conjugated anti-IFN-γ (Pharmingen). ..

Incubation:

Article Title: Induction of Cell-Mediated Immunity to Staphylococcus aureus in the Mouse Mammary Gland by Local Immunization with a Live Attenuated Mutant
Article Snippet: Cytokine production by different lymphocyte subpopulations was evaluated at the single-cell level by intracellular staining by using the Citofix/Cytoperm Plus kit (Pharmingen) according to the manufacturer's instructions. .. Briefly, cultured cells or freshly isolated cells from mice challenged with wt-S. aureus were incubated consecutively with monensin, Cytofix/Cytoperm solution (Pharmingen), and FITC-conjugated anti-IFN-γ (Pharmingen). ..

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    Pharmingen monensin
    Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10 7 spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4 −/− (D), CD8 −/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and <t>monensin</t> for 4 h. Cultured cells were then labeled for CD4 + (A and B) or CD8 + (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P
    Monensin, supplied by Pharmingen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monensin/product/Pharmingen
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2021-03
    86/100 stars
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    Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10 7 spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4 −/− (D), CD8 −/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for CD4 + (A and B) or CD8 + (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P

    Journal: Infection and Immunity

    Article Title: Lack of CD4+ T Cells Does Not Affect Induction of CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

    doi:

    Figure Lengend Snippet: Detection of cytokine production by intracellular staining. Five- to six-week-old wild-type C57BL/6 and age-matched knockout mice were infected with 10 7 spores of E. cuniculi as described in Materials and Methods. At day 15 p.i., total splenocytes from CD4 −/− (D), CD8 −/− (B), and wild-type (A and C) infected mice were cultured in vitro with PMA, ionomycin, and monensin for 4 h. Cultured cells were then labeled for CD4 + (A and B) or CD8 + (C and D) T cells before intracellular staining for IFN-γ, IL-4, and IL-10. Values are the mean percentage of cells positive for IFN-γ, IL-4, or IL-10. Error bars represent the SD for four mice per group. Statistical significance was determined using the Student t test (∗, P

    Article Snippet: The cells were cultured at the concentration of 106 cells/well in a 96-well plate and stimulated with phorbol myristate acetate (PMA; 10 ng/ml; Sigma), ionomycin (500 ng/ml; Sigma), and monensin (GolgiStop; 2 μM; Pharmingen).

    Techniques: Staining, Knock-Out, Mouse Assay, Infection, Cell Culture, In Vitro, Labeling

    Intracellular IL-8 is present in the monocytic subpopulation of PBMC exposed to HIV-1. PBMC were treated with monensin (control) along with HIV-1 BaL , HIV-1 BRU , or TNF-α (100 ng/ml). PBMC were harvested after 6 h and analyzed for intracellular IL-8 protein by flow cytometry. (A) Lymphocyte and monocyte subpopulations were gated according to the pattern of forward scatter and side scatter. (B) Background staining in the lymphocytes and monocytes was determined by incubation with phycoerythrin-mouse IgG1 isotype control (mouse IgG-PE) and FITC-mouse IgG2a isotype control (mouse IgG-FITC). The histograms show fluorescence intensity on logarithmic scales along the x axis (FITC) and y axis (phycoerythrin). The percentage of FITC-positive cells is indicated for both the double-positive and single-positive quadrants. (C) Lymphocytes and monocytes were stained with an FITC-conjugated mouse anti-human IL-8 antibody (IL-8-FITC) and either phycoerythrin-conjugated mouse anti-human CD4 (CD4-PE) or phycoerythrin-conjugated mouse anti-human CD14 (CD14-PE), respectively. The percentage of CD4 + and CD14 + cells staining positive for IL-8 in each condition is indicated. Data shown are representative of four independent experiments.

    Journal: Journal of Virology

    Article Title: Interleukin-8 and Growth-Regulated Oncogene Alpha Mediate Angiogenesis in Kaposi's Sarcoma

    doi: 10.1128/JVI.76.22.11570-11583.2002

    Figure Lengend Snippet: Intracellular IL-8 is present in the monocytic subpopulation of PBMC exposed to HIV-1. PBMC were treated with monensin (control) along with HIV-1 BaL , HIV-1 BRU , or TNF-α (100 ng/ml). PBMC were harvested after 6 h and analyzed for intracellular IL-8 protein by flow cytometry. (A) Lymphocyte and monocyte subpopulations were gated according to the pattern of forward scatter and side scatter. (B) Background staining in the lymphocytes and monocytes was determined by incubation with phycoerythrin-mouse IgG1 isotype control (mouse IgG-PE) and FITC-mouse IgG2a isotype control (mouse IgG-FITC). The histograms show fluorescence intensity on logarithmic scales along the x axis (FITC) and y axis (phycoerythrin). The percentage of FITC-positive cells is indicated for both the double-positive and single-positive quadrants. (C) Lymphocytes and monocytes were stained with an FITC-conjugated mouse anti-human IL-8 antibody (IL-8-FITC) and either phycoerythrin-conjugated mouse anti-human CD4 (CD4-PE) or phycoerythrin-conjugated mouse anti-human CD14 (CD14-PE), respectively. The percentage of CD4 + and CD14 + cells staining positive for IL-8 in each condition is indicated. Data shown are representative of four independent experiments.

    Article Snippet: The next day, PBMC were treated for 6 h with monensin (GolgiStop; PharMingen) and incubated with virus or TNF-α as indicated in the figure legends.

    Techniques: Flow Cytometry, Cytometry, Staining, Incubation, Fluorescence

    Intracellular detection of IL-4 and INF-γ in lymphocytes from lung lavage fluid of IL-9–expressing mice. Data shown are generated by three-color flow cytometric analysis of total lung lavage cells and pooled from four IL-9–expressing littermates after short-term culture in the presence of PMA, ionomycin, and monensin. Dot-blots were gated on CD4 + lymphocytes ( A ) or CD8 + lymphocytes ( B ). Number of cells staining for each cytokine are expressed as a percentage of CD4 + or CD8 + cells.

    Journal: The Journal of Experimental Medicine

    Article Title: Expression of Interleukin 9 in the Lungs of Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness

    doi:

    Figure Lengend Snippet: Intracellular detection of IL-4 and INF-γ in lymphocytes from lung lavage fluid of IL-9–expressing mice. Data shown are generated by three-color flow cytometric analysis of total lung lavage cells and pooled from four IL-9–expressing littermates after short-term culture in the presence of PMA, ionomycin, and monensin. Dot-blots were gated on CD4 + lymphocytes ( A ) or CD8 + lymphocytes ( B ). Number of cells staining for each cytokine are expressed as a percentage of CD4 + or CD8 + cells.

    Article Snippet: In brief, cells retrieved by lung lavage as described above were washed and cultured at a concentration of 5 × 106 in the presence of 0.05 μg/ml PMA ( Sigma Chemical Co. , St. Louis, MO) and 0.5 μg/ml ionomycin ( Calbiochem Corp. , La Jolla, CA) for 6 h. After 2 h of incubation, monensin ( PharMingen ) was added to a final concentration of 2 μM.

    Techniques: Expressing, Mouse Assay, Generated, Flow Cytometry, Staining

    FACScan analysis of TNF-α expression in monocytes and lymphocytes. Monocytes and lymphocytes (5 × 10 5 each) were mixed and incubated for 6 h with 100 ng of SEB per ml in the presence of 2 μM monensin. Samples were fixed and then permeabilized and stained with cell-type-specific antibody and analyzed on a FACScan flow cytometer. Negative controls (A and C) were without SEB. Samples were stained with anti-CD3 or anti-CD14 antisera to identify the following: lymphocytes (A), lymphocytes plus SEB (B), monocytes (C), and monocytes plus SEB (D).

    Journal: Infection and Immunity

    Article Title: Production of Tumor Necrosis Factor Alpha in Human T Lymphocytes by Staphylococcal Enterotoxin B Correlates with Toxin-Induced Proliferation and Is Regulated through Protein Kinase C

    doi:

    Figure Lengend Snippet: FACScan analysis of TNF-α expression in monocytes and lymphocytes. Monocytes and lymphocytes (5 × 10 5 each) were mixed and incubated for 6 h with 100 ng of SEB per ml in the presence of 2 μM monensin. Samples were fixed and then permeabilized and stained with cell-type-specific antibody and analyzed on a FACScan flow cytometer. Negative controls (A and C) were without SEB. Samples were stained with anti-CD3 or anti-CD14 antisera to identify the following: lymphocytes (A), lymphocytes plus SEB (B), monocytes (C), and monocytes plus SEB (D).

    Article Snippet: FITC–anti-human TNF-α monoclonal antibody (MAb) and monensin were from PharMingen (San Diego, Calif.).

    Techniques: Expressing, Incubation, Staining, Flow Cytometry, Cytometry