m2 melanocyte growth medium  (PromoCell)

Journal: eBioMedicine

Article Title: Directed-complement killing of Pseudomonas aeruginosa protects against lethal pneumonia

doi: 10.1016/j.ebiom.2025.105926

Figure Lengend Snippet: CoMiX-Fc enhances significantly phagocytosis of P. aeruginosa by PBMCs-derived M1 macrophages and more importantly by Neutrophils-Like Cells, nevertheless, both CoMiX slightly improve NLCs-dependent antimicrobial activity against the bacteria. PMA-activated M1 macrophages, from four different healthy donors, were co-incubated with pHrodo-stained P. aeruginosa (PAO1) at a 12:1 bacteria-to-cell ratio with 10% NHS and in the presence or absence of 15 μg/mL CoMiX-Fc, CoMiX-FHR1 or CoMiX-irrelevant. Engulfment of bacteria was assessed after 30 min (left panels) and 1 h (right panels) by real-time Incucyte® microscope (a–c) . The percentage of pHrodo positive cells (a) and the intensity of pHrodo rationalised over the surface of cells and calculated as integrated intensity (b) were recorded. Data are presented as the mean values ± SEM. Results correspond to two independent experiments with macrophages from 4 healthy donors (three technical replicates per donor). Statistical analysis was performed using paired one-way ANOVA (to smooth inter-donor variability), followed by Tukey's post-hoc test: ∗p < 0.05; ∗∗p < 0.01. (c) Representative incucyte images for the phagocytosis induced by serum and CoMiX-Fc over time. (d) Fluorescence microscopy image obtained on a wide field Axio Observer Z1, and treated on ImageJ of phagocytosed P. aeruginosa bacteria by PBMCs-derived M1 macrophages in presence of 10% serum and 15 μg/mL CoMiX-Fc. Red = wheat germ agglutinin Alexa-647, staining carbohydates residues of macrophages membranes; green = CellTrace™ CFSE stained P. aeruginosa bacteria. Scale bar = 25 μm. P. aeruginosa PAO1 strain and PAO1-GFP strain were co-cultured with NLCs at a 10:1 bacteria-to-cell ratio, treated with 2% NHS and CoMiX (15 μg/mL) and incubated for 20 min at 37 °C under agitation (200 rpm). After washes and treatment with gentamicin to eliminate non-phagocytosed bacteria from the cells samples were (1) fixed and read by flow cytometry. (e) Gating of NLCs with FSC and SSC to eliminate cell debris and free bacteria from the analysis (left graph). Representative histogram describing the total population of NLCs, and composed of GFP negative cells and GFP positive cells (right graph). To assess phagocytosis, the percentage of GFP positive cells (f) and the mean fluorescence (g) were acquired. Data are presented as the mean values ± SEM. Results correspond to three pooled experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗∗∗∗p < 0.0001. (h) After washes and treatment with gentamicin, cells were also (2) lysed by Triton X-100, diluted in PBS and plated onto petri dishes. CFUs, corresponding to phagocytosed bacteria were counted in duplicates. Data are presented as the mean values ± SEM. Results correspond to three pooled independent experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗∗∗∗p < 0.0001. To assess NLCs-dependent killing, P. aeruginosa PAO1 strain at a MOI of 2:1 was co-cultured with NLCs, treated with 10% NHS and CoMiX (15 μg/mL) for 1 h, plated on petri dishes to count the final bacterial CFUs in duplicates (i) . Conditions which were not co-cultured with NLCs were used as control (100% cell survival). Results are expressed as surviving bacteria compared to bacterial growth under the same conditions in the absence of NLCs. Data are presented as the mean values ± SEM. Results correspond to three pooled independent experiments (2 replicates per experiment). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test: ∗p < 0.05; ∗∗p < 0.01.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from healthy donors (Luxembourg Red Cross, MAN_SCE_24_008) were isolated from buffy coats and differentiated into M1 macrophages using and following the manufacturer's instructions of PromoCell macrophage generation medium (PromoCell, Germany).

Techniques: Derivative Assay, Activity Assay, Bacteria, Incubation, Staining, Microscopy, Fluorescence, Cell Culture, Flow Cytometry, Control