Journal: PLOS Pathogens

Article Title: Adenovirus phagocytosis by neutrophils triggers a pro-inflammatory response

doi: 10.1371/journal.ppat.1013504

Figure Lengend Snippet: (A) Ad cytotoxicity assessed via Sytox Green assay. PMNs were incubated with HBSS, Triton-X-100, human serum (HS) or HS-opsonized Ads (MOI 10 4 vp/cell) in the presence of the cell-impermeant fluorescent probe Sytox Green probe, a cell-impermeant DNA fluorescent probe. Cell fluorescence was measured with a plate reader. The curves represent the percentage (± SEM, 5 independent experiments) of PMN death ( i.e., PMN fluorescence relative to Triton-X-100-lysed PMN fluorescence multiplied by 100) at different incubation times. Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests: *, p < 0.05 (comparison with HS). (B) Ad-induced NET (neutrophil extracellular trap) formation. PMNs were incubated with buffer (control), PMA 100 nM (positive control) or HS-opsonized A488-Ad5 or Ad3 (MOI 10 4 vp/cell) for 4h. Then, after cell fixation, cells were permeabilized, immunolabeled for myeloperoxidase (MPO) and stained with DAPI. The images are Z-projections generated from Z-stacks. Three independent experiments were performed. Scale bar = 10 µm.

Article Snippet: For the second method, PMNs were isolated using the MACSxpress Whole Blood Neutrophil Isolation Kit (Miltenyi Biotec, 130-104-434) and then the MACSxpress Erythrocyte Depletion Kit (Miltenyi Biotec, 130-098-196) making it possible to discard the remaining red blood cells.

Techniques: Incubation, Fluorescence, Comparison, Control, Positive Control, Immunolabeling, Staining, Generated

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: Differential leukocyte sensitivity to planktonic S. aureus . Primary Mφs, G-MDSCs, and PMNs were challenged with planktonic S. aureus at a multiplicity of infection (MOI) of 10:1 (bacteria:leukocyte) for the indicated intervals. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine exposure), and MitoSOX Red (mtROS) to quantify ( A ) leukocyte viability, ( B ) early apoptosis, ( C ) late apoptosis/necrosis, and mtROS levels in ( D ) live and ( E ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 6 h ( n = 12 from three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; One-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: Infection, Bacteria, Staining, Incubation

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: S. aureus biofilm elicits distinct leukocyte responses. Primary Mφs, G-MDSCs, and PMNs were exposed to S. aureus biofilm for the indicated intervals. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine), and MitoSOX Red (mtROS) to quantify ( A ) leukocyte viability, ( B ) early apoptosis, ( C ) late apoptosis/necrosis, and mtROS levels in ( D ) live and ( E ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 2 h ( n = 12 from three independent experiments; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: Staining, Incubation

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: Leukocyte mtROS production has minimal impact on cell death. Primary Mφs, G-MDSCs, and PMNs were pre-treated with MitoTEMPO for 1 h and co-cultured with the indicated strains of S. aureus biofilm or planktonic bacteria for 2 h. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine), and MitoSOX Red (mtROS) to quantify ( A and F ) leukocyte viability, ( B and G ) early apoptosis, ( C and H ) late apoptosis/necrosis, and mtROS levels in ( D and I ) live and ( E and J ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 2 h ( n = 9 from three independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: Cell Culture, Bacteria, Staining, Incubation

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: S. aureus toxins elicit minimal effects on leukocytes during biofilm co-culture. Primary Mφs, G-MDSCs, and PMNs were co-cultured with WT and various S. aureus mutant biofilms for 2 h. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine), and MitoSOX Red (mtROS) to quantify ( A ) leukocyte viability, ( B ) early apoptosis, ( C ) late apoptosis/necrosis, and mtROS levels in ( D ) live and ( E ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 2 h (WT and Unstim. n = 20; Δ hla /Δ lukAB and Δ psmα1-4 /Δ hld n = 12 from three independent experiments; Δ agr n = 8 from two independent experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: Co-Culture Assay, Cell Culture, Mutagenesis, Staining, Incubation

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: Direct contact with S. aureus biofilm is critical to induce leukocyte cytotoxicity. Primary Mφs, G-MDSCs, and PMNs were co-cultured with S. aureus biofilm either directly or separated by Transwell inserts for the indicated intervals. Leukocytes were stained with anti-CD45, Zombie NIR (viability), Apotracker Green (phosphatidylserine), and MitoSOX Red (mtROS) to quantify ( A ) leukocyte viability, ( B ) early apoptosis, ( C ) late apoptosis/necrosis, and mtROS levels in ( D ) live and ( E ) early apoptotic cells. Unstimulated leukocytes were incubated in medium for 30 min or 2 h, as indicated ( n = 9 from three independent experiments; *, P < 0.05; **, P < 0.01; ****, P < 0.0001; one-way ANOVA with Dunnett’s multiple correction between cell types).

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: Cell Culture, Staining, Incubation

Journal: Infection and Immunity

Article Title: Differential sensitivity of leukocyte populations to Staphylococcus aureus biofilm

doi: 10.1128/iai.00654-25

Figure Lengend Snippet: S. aureus -mediated leukocyte death is growth state- and contact-dependent. Mφs, G-MDSCs, and PMNs are the predominant immune cell infiltrates in S. aureus PJI. Planktonically grown S. aureus induced greater cell death in G-MDSCs, whereas Mφs were extremely sensitive to direct biofilm contact. This differential response may account for the large number of granulocytes, most notably anti-inflammatory G-MDSCs, and the minor Mφ infiltrate associated with PJI. Physical separation of all leukocyte populations from the biofilm negated cytotoxicity. Figure created in BioRender. Brandquist, N. (2025) https://BioRender.com/we51cxn.

Article Snippet: For generating primary Mφs, G-MDSCs, and PMNs, C57BL/6J mice (RRID: IMSR_JAX:000664) were euthanized by an overdose of inhaled isoflurane followed by cervical dislocation to collect bone marrow as previously described ( , ).

Techniques: