Journal:

Article Title: Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro

doi: 10.1128/IAI.69.2.665-672.2001

Figure Lengend Snippet: Expression of CD80, CD86, and CD40 on monocytes and macrophages. Three different populations of monocytes/macrophages were assessed for expression of CD80 (A, D, and G), CD86 (B, E, and H), and CD40 (C, F, and I) by flow cytometry before or after culture. (A to C) Expression on freshly isolated CD14+ PBMC. (D to F) Expression on MHC class II+ adherent cells (macrophages [see Materials and Methods]) for cells exposed (gray line) or not exposed (black line) to L. major during the last 24 h of 7-day cultures. Similar results were obtained for macrophages cultured for a total of 14 days. (G to I) Expression on MHC class II+ adherent cells exposed (gray line) or not exposed (black line) to L. major and cultured with PBL for an additional 7 days (14-day total culture). In all panels, shaded histograms represent nonspecific fluorescence of cells stained with an isotype control antibody. The data presented are representative of five individual donors.

Article Snippet: The following reagents were used for flow cytometry: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 (clone TUK4, mouse IgG2a; Caltag, Burlingame, Calif.); phycoerythrin (PE)-labeled anti-human CD80 (clone L307.4, mouse IgG1; PharMingen); PE- and FITC-labeled anti-human CD86 (clone 2331, mouse IgG1; PharMingen); CyChrome-labeled anti-human CD40 (clone 5C3, mouse IgG1; PharMingen); FITC-labeled anti human HLA DR, DP, and DQ (clone TU 39, mouse IgG2a; PharMingen); and appropriately labeled irrelevant isotype-matched control antibodies from the same suppliers.

Techniques: Expressing, Flow Cytometry, Isolation, Cell Culture, Fluorescence, Staining

Journal:

Article Title: Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro

doi: 10.1128/IAI.69.2.665-672.2001

Figure Lengend Snippet: Blockade of CD40-CD154 interaction reduces IFN-γ and IL-5 production after primary and secondary stimulation. PBL were stimulated with autologous L. major-infected adherent cells (macrophages) in the presence or absence of anti-CD154 or an isotype control antibody (mouse IgG1). After 7 days, supernatants were harvested and concentrations of IFN-γ (A) and IL-5 (B) were determined by ELISA. The blast cells from these cultures were isolated and restimulated using autologous L. major-infected adherent cells. After 48 h, supernatants were harvested and concentrations of IFN-γ (C) and IL-5 (D) were determined. The data for individual donors (●) and means (bars) are presented with background cytokine levels subtracted. Background equaled the concentration of cytokine when PBL were stimulated with uninfected adherent cells and ranged <10 to 270 pg/ml for IFN-γ and <2.5 to 5.5 pg/ml for IL-5. The concentrations of IFN-γ and IL-5 observed in cultures treated with isotype control antibody were equivalent to those in cultures with no added antibody (data not shown). ∗, statistically different (P < 0.05).

Article Snippet: The following reagents were used for flow cytometry: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 (clone TUK4, mouse IgG2a; Caltag, Burlingame, Calif.); phycoerythrin (PE)-labeled anti-human CD80 (clone L307.4, mouse IgG1; PharMingen); PE- and FITC-labeled anti-human CD86 (clone 2331, mouse IgG1; PharMingen); CyChrome-labeled anti-human CD40 (clone 5C3, mouse IgG1; PharMingen); FITC-labeled anti human HLA DR, DP, and DQ (clone TU 39, mouse IgG2a; PharMingen); and appropriately labeled irrelevant isotype-matched control antibodies from the same suppliers.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay

Journal:

Article Title: Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro

doi: 10.1128/IAI.69.2.665-672.2001

Figure Lengend Snippet: Effects of CD40-CD154 blockade on IL-10 and IL-12 levels after primary stimulation. PBL were stimulated with autologous L. major-infected adherent cells (macrophages) in the presence or absence of anti-CD154 or an isotype control antibody (mouse IgG1). After 7 days, supernatants were harvested and concentrations of IL-10 (A) and IL-12 (B) were determined by ELISA. The data for individual donors (●) and means (bars) are presented with background cytokine levels subtracted. Background equaled the concentration of cytokine when PBL were stimulated with uninfected adherent cells and ranged 10 to 30 pg/ml for both IL-10 and IL-12. The concentrations of IL-10 and IL-12 observed in cultures treated with isotype control antibody were equivalent to those in cultures with no added antibody (data not shown). ∗, statistically different (P < 0.05).

Article Snippet: The following reagents were used for flow cytometry: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 (clone TUK4, mouse IgG2a; Caltag, Burlingame, Calif.); phycoerythrin (PE)-labeled anti-human CD80 (clone L307.4, mouse IgG1; PharMingen); PE- and FITC-labeled anti-human CD86 (clone 2331, mouse IgG1; PharMingen); CyChrome-labeled anti-human CD40 (clone 5C3, mouse IgG1; PharMingen); FITC-labeled anti human HLA DR, DP, and DQ (clone TU 39, mouse IgG2a; PharMingen); and appropriately labeled irrelevant isotype-matched control antibodies from the same suppliers.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal:

Article Title: Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro

doi: 10.1128/IAI.69.2.665-672.2001

Figure Lengend Snippet: B7 blockade reduces IFN-γ and IL-5 production after primary and secondary stimulation. PBL were stimulated with autologous L. major-infected adherent cells (macrophages) in the presence or absence of anti-CD80, anti-CD86, both antibodies, or an isotype control antibody (mouse IgG1). After 7 days, supernatants were harvested and concentrations of IFN-γ (A) and IL-5 (B) were determined by ELISA. The blast cells from these cultures were isolated and restimulated using autologous L. major-infected adherent cells. After 48 h, supernatants were harvested and concentrations of IFN-γ (C) and IL-5 (D) were determined. The data for individual donors (●) and means (bars [geometric for IFN-γ; arithmetic for IL-5]) are presented with background cytokine levels subtracted. Background equaled the concentration of cytokine when PBL were stimulated with uninfected macrophages and ranged from <10 to 270 pg/ml for IFN-γ or <2.0 to 5.5 pg/ml for IL-5. The concentrations of IFN-γ and IL-5 observed in cultures treated with isotype control antibody were equivalent to those in cultures with no added antibody (data not shown). Means with different letters are statistically different (P < 0.05).

Article Snippet: The following reagents were used for flow cytometry: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 (clone TUK4, mouse IgG2a; Caltag, Burlingame, Calif.); phycoerythrin (PE)-labeled anti-human CD80 (clone L307.4, mouse IgG1; PharMingen); PE- and FITC-labeled anti-human CD86 (clone 2331, mouse IgG1; PharMingen); CyChrome-labeled anti-human CD40 (clone 5C3, mouse IgG1; PharMingen); FITC-labeled anti human HLA DR, DP, and DQ (clone TU 39, mouse IgG2a; PharMingen); and appropriately labeled irrelevant isotype-matched control antibodies from the same suppliers.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Isolation, Concentration Assay

Journal:

Article Title: Influence of Costimulatory Molecules on Immune Response to Leishmania major by Human Cells In Vitro

doi: 10.1128/IAI.69.2.665-672.2001

Figure Lengend Snippet: Effects of B7 blockade on IL-10 and IL-12 levels after primary stimulation. PBL were stimulated with autologous L. major-infected adherent cells (macrophages) in the presence or absence of anti-CD80, anti-CD86, both antibodies, or an isotype control antibody (mouse IgG1). After 7 days, supernatants were harvested and concentrations of IL-10 (A) and IL-12 (B) were determined by ELISA. The data for individual donors (●) and means (bars) are presented with background cytokine levels subtracted. Background equaled the concentration of cytokine when PBL were stimulated with uninfected adherent cells and ranged from <10 to 30 pg/ml for both IL-10 and IL-12. The concentrations of IL-10 and IL-12 observed in cultures treated with isotype control antibody were equivalent to those in cultures with no added antibody (data not shown). Means with different letters are statistically different (P < 0.05).

Article Snippet: The following reagents were used for flow cytometry: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 (clone TUK4, mouse IgG2a; Caltag, Burlingame, Calif.); phycoerythrin (PE)-labeled anti-human CD80 (clone L307.4, mouse IgG1; PharMingen); PE- and FITC-labeled anti-human CD86 (clone 2331, mouse IgG1; PharMingen); CyChrome-labeled anti-human CD40 (clone 5C3, mouse IgG1; PharMingen); FITC-labeled anti human HLA DR, DP, and DQ (clone TU 39, mouse IgG2a; PharMingen); and appropriately labeled irrelevant isotype-matched control antibodies from the same suppliers.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: AIP56, an AB toxin secreted by Photobacterium damselae subsp. piscicida , has tropism for myeloid cells

doi: 10.3389/fimmu.2024.1527088

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: Human leukocytes , Mouse anti-human CD14 , PE-Cy7 , 61D3 , eBiosciences/25-0149-42.

Techniques: Cytometry

Journal: PLoS Pathogens

Article Title: Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

doi: 10.1371/journal.ppat.1002814

Figure Lengend Snippet: A) Peripheral blood mononuclear cells (PBMC), purified CD3 + T-cells (CD3 + CD14 − ) or co-cultures of purified CD3 + T-cells with 10% purified CD14 + monocytes (CD3+ CD14+) were mock-infected (MOI = 0, white bars) or challenged with D39 Streptococcus pneumoniae (MOI = 50, black bars) for 16 h and accumulation of hypodiploid DNA (% Sub G0/1) measured in CD3 + T-cells, n = 4. B) caspase 3 activation in CD3 + T-cells under the same condition as A), n = 4. C) Representative western blot probed for Bid, Bim and actin from CD3 + T-cells isolated from PBMC or CD3 enriched cultures following bacterial challenge as in A), D) densitometry summarizes the fold change in Bid compared to the mock infected (MI), derived from three separate experiments. E) % Sub G0/1 purified CD3 + T-cells in co-cultures containing 10% or 20% of purified CD14 + monocytes under the same conditions as A), n = 4 or in F) co-cultures of CD3 + T-cells and purified CD14 + monocytes with (CD14 + /CD16 − ) or without (CD14 + /CD16 + ) CD16 + monocyte depletion, under the same conditions as A), n = 4, ns (not significant) * p<0.05, ** p<0.01, *** p<0.001; statistical analysis by ANOVA.

Article Snippet: Cell surface marker expression was with 1 μg/ml mouse anti-human anti- CD14 (61D3) phycoerythrin (PE), (eBioscience), anti-CD3 (SK7) fluorescein isothiocyanate (FITC), (BD Pharmingen), anti-CD161 (HP-3G10) allophycocyanin (APC), (eBioscience), anti-CD4 (CSK3) peridinin chlorophyll protein (PerCP) and anti-CD19 (HIB19) FITC (BD Pharmingen) with appropriate isotype controls.

Techniques: Purification, Infection, Activation Assay, Western Blot, Isolation, Derivative Assay

Journal: PLoS Pathogens

Article Title: Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

doi: 10.1371/journal.ppat.1002814

Figure Lengend Snippet: A) Mean caspase 8 relative luminescence units (Caspase 8 RLU) in CD3 + T-cells from peripheral blood mononuclear cells (PBMC), purified CD3 + T-cells (CD3+ CD14−) or co-cultures of purified CD3 + T-cells with 10% purified CD14 + monocytes (CD3+ CD14+) 6 h following mock-infection (multiplicity of infection (MOI) = 0) or challenge with D39 Streptococcus pneumoniae (MOI = 50), n = 5. B) Representative western blot probed for Bid, Bim and actin from CD3 + T-cells purified from PBMC 16 h following mock-infection (D39−) or challenge with D39 at MOI = 0.1 (D39+) in the presence of isotype control (Isotype+) or ZB4 neutralizing anti-Fas antibody (Anti-Fas+) added at 1 µg/ml. C) Densitometry summarises the fold change in Bid compared to the mock infected (MI) cells from three separate experiments with different donors. D) Cytosolic and membrane fractions were also probed for cytochrome c, actin and Cox-4, and E) densitometry was performed, n = 4. F) Hypodiploid DNA accumulation (Sub G0/1) was measured in CD3 + T-cells in PBMC cultured under the same conditions as B), n = 4. G) The percentage of Annexin V + CD3 + T-cells in PBMC cultured under the same condition as in B), n = 3, * p<0.05; statistical analysis by ANOVA or t-test.

Article Snippet: Cell surface marker expression was with 1 μg/ml mouse anti-human anti- CD14 (61D3) phycoerythrin (PE), (eBioscience), anti-CD3 (SK7) fluorescein isothiocyanate (FITC), (BD Pharmingen), anti-CD161 (HP-3G10) allophycocyanin (APC), (eBioscience), anti-CD4 (CSK3) peridinin chlorophyll protein (PerCP) and anti-CD19 (HIB19) FITC (BD Pharmingen) with appropriate isotype controls.

Techniques: Purification, Infection, Western Blot, Control, Membrane, Cell Culture