human monocyte cell line thp  (CLS Cell Lines Service GmbH)

Journal: ACS Infectious Diseases

Article Title: Recombinant Human IgG1 Enhances Complement-Mediated Bacteriolysis and Macrophage Phagocytosis of Pseudomonas aeruginosa via Targeting Cell Surface Polysaccharides

doi: 10.1021/acsinfecdis.5c01032

Figure Lengend Snippet: yPgi3G4 IgG1-mediated phagocytosis of P. aeruginosa by THP-1 monocytes. Confocal microscopy images were taken with the 100× inverted oil immersion lens of a Nikon A1plus confocal microscope. The scale bars represented 20 μm. (A) Mature THP-1 cells after challenging with P. aeruginosa in the presence of yPgi3G4 IgG1 (A1–A3), irrelevant antibacterial IgG1 (A4–A6), or without IgG (A7–A9). The nuclei of THP-1 cells were stained blue by DAPI. P. aeruginosa was stained green by yPgi3G4 scFv, followed by Dylight 488-labeled antihexa-histidine mAb. (B) Mature THP-1 cells after incubation with P. aeruginosa in the presence of yPgi3G4 IgG1 (B1–B8) or without IgG (B9–B16). In B1, an example of a phagosome dot at the surface of THP-1 was marked with a white arrow. In this experiment, yPgi3G4 IgG1 was stained pink by DyLight 650-tagged goat F­(ab’)­2 antihuman IgG-Fc. Actin of THP-1 cells was stained red by Texas Red-X phalloidin.

Article Snippet: A fresh overnight P. aeruginosa culture in LB broth (1–2 × 10 9 CFU/mL) was washed with 1xPBS for once, resuspended in equal volume of 1xPBS, and serially diluted in antibiotic-free RPMI medium so that 8 × 10 5 cells of untreated undifferentiated THP-1 monocytes (CLS# 300356–1714SF, Germany) were exposed to 4.5 × 10 4 or 4.5 × 10 3 or 4.5 × 10 2 or 45 CFU of P. aeruginosa in 300 μL reaction volume.

Techniques: Confocal Microscopy, Microscopy, Staining, Labeling, Incubation

Journal: ACS Infectious Diseases

Article Title: Recombinant Human IgG1 Enhances Complement-Mediated Bacteriolysis and Macrophage Phagocytosis of Pseudomonas aeruginosa via Targeting Cell Surface Polysaccharides

doi: 10.1021/acsinfecdis.5c01032

Figure Lengend Snippet: Caveolin-1 participates in phagocytic clearance of P. aeruginosa . Confocal microscopy images (A–H) taken with 60× upright oil immersion lens of a Nikon Eclipse microscope. The scale bars represented 10 μm. The nuclei of THP-1 cells were stained blue by DAPI. P. aeruginosa cells were detected by yPgi3G4 scFv and stained green with Dylight 488-labeled antihexa-histidine mAb. Caveolin-1 of THP-1 monocytes was detected with anticaveolin-1 mAb and stained red by Alexa Fluor 594-labeled goat antimouse IgG. IgG1 in 30 min incubation samples was stained pink by DyLight 650-goat F­(ab’)­2 antihuman IgG-Fc. (A) VitD3-differentiated THP-1 cells at 30 min after challenging with P. aeruginosa and (A1–A4) no IgG, (A5–A8) irrelevant antibacterial IgG1, and (A9–A12) yPgi3G4 IgG1. ( B ) VitD3-differentiated THP-1 cells at 24 h after challenging with P. aeruginosa and (B1–B4) no IgG, (B5–B8) irrelevant antibacterial IgG1, and (B9–B12) yPgi3G4 IgG1. In B9, the arm-like THP-1 cell surface projections were indicated with white arrows. (C–H) Inset of A4, A8, A12, B4, B8, and B12. (I) The Dylight 488 (green) intensity inside THP-1 cells was analyzed using ImageJ 1.54g software (NIH, U.S.A.). (J) IL-8 and IL1-β production by THP-1 monocytes at 24 h. Each bar represented the average value ( n = 2–3) and error bars represented the standard error of the mean (SEM). (K) IL1-β production by THP-1 monocytes within 24 h. Each bar represented the average value ( n = 2) and error bars represented the SEM. Statistical analysis in (I) and (K) was performed by Two-way ANOVA Tukey’s multiple comparisons test. The asterisks (**) and (****) indicated P value 0.0021 and <0.0001, respectively. (L) The turbidity and the CFU/mL of P. aeruginosa in the supernatant of THP-1 cells infected with indicated inoculum (at the top of the graph) and treatment (at the bottom of the graph) for 24 h were reported ( n = 2). The fresh RPMI medium and the uninfected THP-1 cells were used as negative controls. OD values were plotted on the left y -axis and each bar represented the average OD and error bars represented the standard error of the mean. The CFU/mL of P. aeruginosa was plotted on the right y -axis and the results were shown as black dots with error bars. Statistical analysis was performed by Ordinary One-way ANOVA Tukey’s multiple comparisons test with P value style of 0.0332 (*), 0.0021 (**), and 0.0002 (***).

Article Snippet: A fresh overnight P. aeruginosa culture in LB broth (1–2 × 10 9 CFU/mL) was washed with 1xPBS for once, resuspended in equal volume of 1xPBS, and serially diluted in antibiotic-free RPMI medium so that 8 × 10 5 cells of untreated undifferentiated THP-1 monocytes (CLS# 300356–1714SF, Germany) were exposed to 4.5 × 10 4 or 4.5 × 10 3 or 4.5 × 10 2 or 45 CFU of P. aeruginosa in 300 μL reaction volume.

Techniques: Confocal Microscopy, Microscopy, Staining, Labeling, Incubation, Software, Infection