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