growth medium rpmi 1640  (ATCC)

Journal: mBio

Article Title: Macrophage phagocytosis of human norovirus-infected cells in an ex vivo human enteroid-macrophage coculture model

doi: 10.1128/mbio.01180-25

Figure Lengend Snippet: HIEs cocultured with all macrophages subtypes remain structurally and morphologically intact. (A) Expression of CD14, CD68, CD80, and CD206 in macrophages relative to monocytes derived from five donors. Dotted line indicates 100% in mean fluorescence intensity (MFI). Gray, red, and green bars indicate M0, M1, and M2 macrophages, respectively. Data are presented as mean ± SD. Statistical significance was determined using Dunnett’s multiple comparisons test, comparing the relative expression between macrophage phenotypes. ** P < 0.01, **** P < 0.0001. The gating stratgey and superimposed plots displaying macrophage heterogeneity from individual donors are shown in and . ( B ) H&E and immunofluorescence images showing adherent M0, M1, and M2 macrophages (CD68; red) to differentiated HIEs (CK8; white) at 1 day post coculture indicated by arrows. Scale bar = 20 µm. ( C ) Number of adherent macrophages per centimeter of HIE monolayer. Measurement of ( D ) epithelial cell height and ( E ) barrier integrity in HIE-macrophage cocultures relative to HIEs alone. Data are presented as mean ± SD and compiled from five donors. Each color corresponds to macrophages derived from the PBMCs of a single donor. Area-normalized TEER values for pre- and post-coculture conditions are shown in .

Article Snippet: The macrophage growth medium consists of RPMI-1640 medium ATCC modification (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1× MEM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 55 μM β-mercaptoethanol (Gibco), and 100 ng/mL M-CSF (PeproTech).

Techniques: Expressing, Derivative Assay, Fluorescence, Immunofluorescence

Journal: mBio

Article Title: Macrophage phagocytosis of human norovirus-infected cells in an ex vivo human enteroid-macrophage coculture model

doi: 10.1128/mbio.01180-25

Figure Lengend Snippet: HuNoV replication is not altered in the presence of macrophages. (A) A schematic diagram showing the compartmentalization (apical, membrane, and basolateral) of HIE-macrophage cocultures in Transwells. HIEs and HIEs cocultured with M0, M1, or M2 macrophages were inoculated with HuNoV at 2.5 × 10 5 genome equivalents per well. Virus replication was evaluated by assessing viral RNA levels in ( B ) apical, ( C ) membrane, and ( D ) basolateral compartments at 2 h and 24 h post infection. Data are presented as means ± SD. Each color corresponds to monocyte-derived macrophages derived from the PBMCs of a single donor used for one coculture experiment. Macrophages derived from five donors were tested.

Article Snippet: The macrophage growth medium consists of RPMI-1640 medium ATCC modification (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1× MEM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 55 μM β-mercaptoethanol (Gibco), and 100 ng/mL M-CSF (PeproTech).

Techniques: Membrane, Virus, Infection, Derivative Assay

Journal: mBio

Article Title: Macrophage phagocytosis of human norovirus-infected cells in an ex vivo human enteroid-macrophage coculture model

doi: 10.1128/mbio.01180-25

Figure Lengend Snippet: Phagocytosis of HuNoV-infected epithelial cells by all macrophage subtypes. Immunofluorescence labelling of HuNoV structural antigen (VP1; green) in epithelial cells (CK8; white) and macrophages (CD68; red) with ( A ) top views and ( B ) orthogonal views. Regions containing CK8/VP1/CD68-positive cells are highlighted with dotted outlines, and orthogonal views are illustrated from the boxed areas. ( C ) Percent of HuNoV-infected cells in infected HIEs and HIE-macrophage cocultures at 24 hpi. Data are represented as mean ± SD and compiled from three experiments. Each color corresponds to monocyte-derived macrophages derived from the PBMCs of a single donor used for one coculture experiment. Statistical significance in the number of CD68/CK8 double-positive cells with VP1 positivity was assessed in infected HIEs cocultured with either M0 or M1 macrophages using Dunnett’s multiple comparisons test; * P < 0.05.

Article Snippet: The macrophage growth medium consists of RPMI-1640 medium ATCC modification (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1× MEM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 55 μM β-mercaptoethanol (Gibco), and 100 ng/mL M-CSF (PeproTech).

Techniques: Infection, Immunofluorescence, Derivative Assay

Journal: mBio

Article Title: Macrophage phagocytosis of human norovirus-infected cells in an ex vivo human enteroid-macrophage coculture model

doi: 10.1128/mbio.01180-25

Figure Lengend Snippet: The presence of both naïve and activated macrophages induces bilateral secretion of cytokines in differentiated HIEs. Levels of 27 cytokines measured in apical ( A, B, C, D ) and basolateral ( E, F, G, H ) compartments of HIEs (white) and HIEs cocultured with M0 (gray), M1 (red), and M2 (green) macrophages. Cytokines are grouped into minimum, low, medium, and high panels based on similar detected levels for better visualization of data. Data are represented as mean ± SD and compiled from four donors. Each dot represents PBMC-derived macrophages used in HIE-macrophage coculture. Statistical significance was determined using a one-way ANOVA with Kruskal-Wallis test; * P < 0.05 and ** P < 0.01.

Article Snippet: The macrophage growth medium consists of RPMI-1640 medium ATCC modification (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1× MEM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 55 μM β-mercaptoethanol (Gibco), and 100 ng/mL M-CSF (PeproTech).

Techniques: Derivative Assay

Journal: mBio

Article Title: Macrophage phagocytosis of human norovirus-infected cells in an ex vivo human enteroid-macrophage coculture model

doi: 10.1128/mbio.01180-25

Figure Lengend Snippet: Chemokines and acute-phase inflammatory mediators were produced apically in HuNoV-infected HIEs cocultured with activated macrophages. Supernatants collected apically and basolaterally from the 24h post HuNoV-infected HIEs and HIE-macrophage cocultures were assessed for cytokine secretion. Twofold increases in the cytokine levels of the HuNoV-infected group normalized to the γ–irradiated HuNoV-infected group are shown. ( A ) A heatmap showing the log 2 fold increases in cytokine levels. The x -axis displays the number of PBMC donors used for HIE-macrophage cocultures and the average value across all donors. Cells with less than a twofold change are depicted as white. Fold increases in the levels of ( B ) GM-CSF and ( C ) TNF-α secreted basolaterally. Dashed line indicates a twofold change. Waterfall plots of Spearman correlation analysis for fold increase in cytokine secreted (D) apically and ( E ) basolaterally to the phagocytosis index, defined by the number of CD68/CK8 double positive cells with VP1 positivity in HIE-macrophage coculture at 24 hpi. This correlation analysis was performed for donors 2, 3, and 4. Cytokines with statistically significant correlation to phagocytosis are labeled in red bars. Spearman r > 0.75, P < 0.05

Article Snippet: The macrophage growth medium consists of RPMI-1640 medium ATCC modification (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 1× MEM non-essential amino acids (Sigma), 1 mM sodium pyruvate (Sigma), 55 μM β-mercaptoethanol (Gibco), and 100 ng/mL M-CSF (PeproTech).

Techniques: Produced, Infection, Irradiation, Labeling