centrifuge ig ic en 21 r  (Orto alresa)

Journal: Frontiers in Immunology

Article Title: Targeting the capsule of Klebsiella pneumoniae with a cationic CR3-binding protein enhances phagocytosis and promotes bacterial clearance and survival in a mouse sepsis model

doi: 10.3389/fimmu.2026.1934439

Figure Lengend Snippet: rPF4 enhances phagocytosis of heat-inactivated and live low-virulence K. pneumoniae by macrophages. (A) Effect of rPF4 on phagocytosis of K. pneumoniae by suspended IC-21 macrophages. Cells were incubated with fluorescein-labeled, heat-inactivated K. pneumoniae (ATCC 13883; capsule type K3) pretreated with 50 µg/ml rPF4 for 30 minutes at 37 °C. After quenching fluorescence with ethidium bromide, phagocytosis was quantified by flow cytometry. Representative histograms from eight independent experiments are shown. (B) Quantification of the effect of different concentrations of rPF4 on phagocytosis of K. pneumoniae by suspended mouse IC-21 macrophages based on flow cytometry results. MFI, mean fluorescence intensity. Data are presented as the mean fold difference in MFI ± standard deviation from 3–8 independent experiments. *p < 0.05, ** p < 0.01, *** p < 0.001, ns, nonsignificant. (C–F) Quantification of phagocytosis of K. pneumoniae by various phagocytes in suspension in the presence of different concentrations of rPF4. Values represent MFI fold-difference means ± S.D. from 3–8 independent experiments. (G) Fluorescein-labeled, heat-inactivated K. pneumoniae were pretreated with 50 µg/ml rPF4 and added to adherent TG-elicited inflammatory macrophages for 60 minutes at 37 °C. Non-phagocytosed bacteria were removed, and phagocytosis was assessed from fluorescent cell images. Representative images of macrophages exposed to rPF4-coated bacteria ( upper panel ) and uncoated control bacteria ( lower panel ) are shown. The scale bars are 10 μm. (H) Representative confocal image showing phagocytosed fluorescein-labeled K. pneumoniae inside a macrophage. Representative confocal image showing phagocytosed fluorescein-labeled K. pneumoniae inside a macrophage with orthogonal YZ and XZ views. The scale bar is 5 μm. The scale bar is 5 μm. (I) Quantification of rPF4-mediated enhancement of K. pneumoniae phagocytosis, expressed as the number of fluorescent bacteria per cell. Phagocytosis was assessed in IC-21 macrophages, mouse inflammatory macrophages, and mouse neutrophil/monocyte populations. Values represent mean ± S.D. from three random fields per condition in three independent experiments ***p < 0.001. (K) Live pHrodo-labeled K. pneumoniae were preincubated with rPF4 and then added to various macrophages in suspension. After 30 min at 37 °C, phagocytosis was measured by flow cytometry. Values represent mean fold differences in MFI ± S.D. from 4–9 independent experiments; ***p < 0.001. (L) Adherent IC-21 macrophages (5×10 5 ) were incubated with 10 6 CFU of K. pneumoniae pretreated with 50 μg/ml rPF4 for 1 h at 37 °C. After phagocytosis, cells were washed, and extracellular bacteria that may have remained on the cell surface were inactivated with gentamicin for 1 h. Macrophages were then either lysed immediately with 0.2% Triton X-100 or cultured in DMEM for 3 and 24 hours. Aliquots of cell lysates were plated on LB agar plates for 16 h at 37 °C, and CFUs were determined. Data are means ± S.D. from 3 experiments. ***p< 0.001.

Article Snippet: The IC-21 murine macrophage cell line, human monocytic U937 and THP-1 cells, and A549 lung epithelial cells were obtained from ATCC and cultured in RPMI supplemented with 10% FBS and antibiotics.

Techniques: Incubation, Labeling, Fluorescence, Flow Cytometry, Standard Deviation, Suspension, Bacteria, Control, Confocal, Cell Culture

Journal: Frontiers in Immunology

Article Title: Targeting the capsule of Klebsiella pneumoniae with a cationic CR3-binding protein enhances phagocytosis and promotes bacterial clearance and survival in a mouse sepsis model

doi: 10.3389/fimmu.2026.1934439

Figure Lengend Snippet: rPF4 enhances phagocytosis of heat-inactivated and live high-virulence K. pneumoniae . (A–D) Heat-inactivated pHrodo-labeled hv Kp (ATCC 43816; capsule type K2) were preincubated with various concentrations of rPF4 for 1 hour at 37 °C and rPF4-coated bacteria were then added to suspended IC-21 macrophages, PMA-activated THP-1 cells, TG-elicited inflammatory mouse macrophages, and mouse peritoneal neutrophils/monocytes for 30 minutes at 37 °C, and phagocytosis was measured by flow cytometry. (E–H) , Live bacteria were labeled with pHrodo, preincubated with rPF4, and then added to various leukocyte populations to assess phagocytosis. Data are presented as means ± S.D. from three separate experiments. ***p< 0.001. (I) The table shows fold changes in phagocytosis at two concentrations of rPF4 (50 and 100 μg/ml) relative to phagocytosis in the absence of rPF4 across various macrophage types.

Article Snippet: The IC-21 murine macrophage cell line, human monocytic U937 and THP-1 cells, and A549 lung epithelial cells were obtained from ATCC and cultured in RPMI supplemented with 10% FBS and antibiotics.

Techniques: Labeling, Bacteria, Flow Cytometry

Journal: Theranostics

Article Title: Toward the clinical translation of safe intravenous long circulating ILNEs contrast agent for CT imaging.

doi: 10.7150/thno.110014

Figure Lengend Snippet: Figure 3. Characterization and stability of ILNEs in various conditions. (A-B) Particle size stability in HP and (C-D) FBS: 10% and 20% (v/v) ILNEs were incubated with (A-B) HP or FBS (C-D) for 48 h. Particle size (A-C) and PDI (B-D) were measured at different time points using DLS. (E) UV-vis absorbance measurements of ILNE1–4, showing the peaks of CrEL and TIPhO at λmax of ~230 nm and ~244 nm, respectively. (F) Viscosity measurements of ILNE1–4, compared with iodixanol stock solution (300 mgI/mL) at 60% (wt/v). (G) Phantom scan of ILNEs: In vitro evaluation of x-ray attenuation properties (in HU) of ILNE1–4 using a calibration curve made by various dilutions of the iodixanol injection as a reference (expressed in mgI/mL). Air and water were used for normalization. Top view of Eppendorf tubes and source data are provided as a Source Data file. (H) CCK-8 cytotoxicity assay of immune cells: Macrophages RAW264.7 and IC21 cells were incubated with ILNE3 for 48 h. Data are presented as the average ± SD (n = 6). (I) Cell viability studies of ILNE components: Two concentrations of CrEL and TIPhO were incubated with RAW 264.7 cells for 24 h.

Article Snippet: Human Dermal Fibroblasts, neonatal (HDFn), RAW 264.7 macrophages and IC21 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Incubation, Viscosity, In Vitro, Injection, CCK-8 Assay, Cytotoxicity Assay