Journal: bioRxiv

Article Title: Beta-2-microglobulin stimulates neutrophil phagocytosis of bacteria and apoptotic cells

doi: 10.1101/2025.10.30.685267

Figure Lengend Snippet: Efferocytosis of apoptotic Jurkat cells labeled with CellTrace™ Violet by CD66b-positive granulocytes stained with CellTrace™ Far Red was examined under various conditions. (A) PMNs were exposed to varying concentrations of β2m or dK58β2m in the presence of apoptotic Jurkat cells. (B) PMNs were treated with apoptotic cells alone or in combination with 50 µg/ml β2m, 50 µg/ml dK58β2m, 100 ng/ml GM-CSF, or combinations thereof. (C-D) PMNs were treated with apoptotic Jurkat cells with 50 µg/ml β2m (C) or dK58β2m (D) combined with 10 or 25% human AB serum. In all experiments, a 4:1 apoptotic cell-to-PMN ratio was used. Treatment with 100 ng/ml GM-CSF served as a positive control, and pre-incubation with 10 µg/ml cytochalasin D (cyto D) served as a negative control. Results are shown as mean ± SD of double-positive PMNs. Sample sizes were n=6 or n=4 (A), n=10 (B), and n=8 or 3 donors (C and D). Group comparisons were analyzed using mixed-effects analysis and Tukey’s multiple comparisons test (A, C, and D) or repeated-measures ANOVA and Tukey’s multiplecomparisons test(B). Statistical significance is indicated by *P < 0.05 and **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: As a control for phagocytic uptake, PMNs were pre-treated with 10 μg/ml cytochalasin D (Thermo Fisher Scientific, #PHZ1063) for 20 min. After the two-hour incubation, the cells were washed in HBSS, and granulocytes were stained with anti-CD66b antibodies (R&D systems, #FAB4246G) for 30 min at 4°C in the dark.

Techniques: Labeling, Staining, Positive Control, Incubation, Negative Control

Journal: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: Bone tissues surgically removed from PJI patient with S. aureus osteomyelitis were processed for histology and immunohistochemistry. (A) Representative 100x image (bar =100 μm) of a H&E-stained section is shown to illustrate the inflammatory cells within the region of interest (box). ( B-D) Parallel histology sections containing the region of interest were immunostained with labelled antibodies against CD3, PD1, S. aureus, TIM-3 (green), LAG-3, and CD66b, counter stained with DAPI, and representative fluorescent microscopy images are shown at 200x (bar = 100 μm). ( B ) Note CD3 + /PD1 + T cells detected in areas of S. aureus infection (white arrows). (C) Note CD3 + /TIM-3 + (white arrows) and CD3 + /LAG-3 + (yellow arrows) T cells at the site of S. aureus infection. (D) Note TIM-3 + /CD66b+ neutrophils at the site of infection (white arrows).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Immunohistochemistry, Staining, Microscopy, Infection