rneasy kit iso modc  (Qiagen)

Journal: bioRxiv

Article Title: Modulating Innate Immune Responses to Curli Fibers Through Protein Engineering

doi: 10.64898/2026.03.23.713613

Figure Lengend Snippet: (A) Schematic of MoDC generation. CD14⁺/CD16⁻ classical monocytes were isolated from a leukopak by immunomagnetic enrichment and differentiated into MoDCs using established cytokine-driven protocols prior to stimulation with purified curli fibers . (B) IL-8 secretion measured by ELISA following 24 h exposure to LPS (the canonical MoDC maturation stimulus), CU-T12-9 (TLR2/TLR1 positive control), Curli (WT), Curli-SSL3, or Curli-S6E11. Values are reported in pg/mL with background subtracted (untreated MoDC baseline set to 0). Statistical comparison between Curli (WT) and Curli-SSL3 was performed using Welch’s t-test. (C-E) RT-qPCR analysis of inflammatory gene expression following 24 h curli exposure: (C) IL-8, (D) IL-6, (E) IL-1β. Data are expressed as fold change relative to untreated MoDCs (set to 1). Statistical comparisons between Curli (WT) and each engineered variant were performed using Welch’s t-tests with Bonferroni correction for multiple pairwise comparisons. Data in panels B-E represent mean ± SD. Statistical significance is indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: To confirm that curli fiber-induced inflammatory responses were mediated primarily through TLR2 signaling, MoDCs were pretreated on day 6 of differentiation with the TLR2 signaling inhibitor TL2-C29 (100 μM; Invivogen, cat. #inh-c29) for 3 hours at 37°C.

Techniques: Isolation, Purification, Enzyme-linked Immunosorbent Assay, Positive Control, Comparison, Quantitative RT-PCR, Gene Expression, Variant Assay

Journal: ImmunoHorizons

Article Title: Direct contact with natural killer cells reprograms monocyte-derived dendritic cells into a tolerogenic phenotype

doi: 10.1093/immhor/vlag007

Figure Lengend Snippet: IL-10 signaling is critical for the tolerogenic effect from the NK-programmed DC. (A) Study design of knocking-out transcription factors and evaluating the resultant DC tolerance effects. (B) IL-10, TNF-α, and IL-6 cytokine production was assayed from LPS-stimulated MoDCs after CRISPR-Cas9 editing. CRISPR-KO MoDC effect on T-cell activation in MLR evaluated by flow cytometry showing (C) CD25 expression on CD4 T cells and CD8 T cells ( n = 3 donors) and (D) evaluated MLR secreted cytokines IL-10, TNF-α, and IFN-γ ( n = 3 donors). Means ± SEM are shown, and statistical analysis is determined by one-way ANOVA Fisher least significant difference test compared to the scramble sgRNA control. * P < .05, ** P < .01, *** P < .001, **** P < .0001. (E) Study design of neutralizing IL-10 during the depletion of MoDCs by anti-DCIR–induced ADCC effect. (F) Cell count and (G, H) MFI of DC co-stimulatory/maturation markers (CD40 and CD44) on surviving CD14 + CD11c + MoDCs in the ADCC assay induced by anti-DCIR antibody in combination with or without anti-IL-10 neutralizing antibody ( n = 3 donors). (I) IL-10R expression and (J) cell viability of the MoDCs differentiated by GM-CSF and IL-4 treatment and matured by CpG and CD40L stimulation, along with indoximod, an IDO1 inhibitor ( n = 4 donors). (K) Schematic summary of the tolerogenesis of DCs from the NK engagement induced by DC-targeting antibody (eg anti-DCIR).

Article Snippet: For the MLR experiment in , PBMCs were isolated from fresh human whole blood and treated with 40 ng/mL human IL-4 and 100 ng/mL human GM-CSF for 3 days to induce MoDC differentiation, followed by CpG ODN2006 (Invivogen, Cat# tlrl-2006) and CD40L (PeproTech, Cat#310-02) to promote MoDC maturation in addition to anti-DCIR antibody to induce antibody-dependent cellular cytotoxicity (ADCC) for 2 to 3 days.

Techniques: CRISPR, Activation Assay, Flow Cytometry, Expressing, Control, Cell Characterization, ADCC Assay

Journal: Viruses

Article Title: Detection of Foodborne Viruses in Dates Using ISO 15216 Methodology

doi: 10.3390/v17020174

Figure Lengend Snippet: MNV recovery from whole Medjool dates and RT-qPCR inhibition.

Article Snippet: The viruses were extracted using the ISO 15216-1:2017 standard protocol version for soft fruit (ISO-modA) combined with the eGene-up RNA extraction process (Biomérieux, Montréal, QC, Canada), or the vegetable version combined with either the eGene-up (ISO-modB) or the RNeasy kit (ISO-modC) (Qiagen, Mississauga, ON, Canada) to extract the viral RNA following the manufacturer’s instructions.

Techniques: Inhibition

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 impairs the function of monocyte-derived Dendritic Cells (A) Alveolar macrophage and dendritic cell populations in bronchoalveolar lavage of patients with lung transplant with and without invasive aspergillosis ( n = 3). (B) Phagocytosis of A. fumigatus conidia (MOI = 1) by dendritic cells untreated, treated with IFNy or FK506 or both for 30 min ( n = 3). (C) Fungal killing of A. fumigatus conidia (MOI = 1) by dendritic cells untreated, treated with IFNy or FK506 or both 6 h post infection, measured by CFUs ( n = 3). (D) Representative ImageStream images of CD83 on moDCs treated with or without IFNy. (E) MFI of CD83 on moDCs untreated, treated with IFNy and/or FK506, infected with A. fumigatus (MOI = 1) or uninfected ( n = 3). (F) Representative ImageStream images of moDCs stained for nucleus (DAPI) and NFATc2 (Cy5), and infected with A. fumigatus (MOI = 1) or uninfected ( n = 3). (G) Timecourse of NFATc2 nuclear translocation of A. fumigatus infected (MOI = 1) moDCs treated with and without FK506 ( n = 3). (H) Time course RCAN-1 expression of Aspergillus infected (MOI = 1) moDCs DCs treated with and without FK506 ( n = 3). ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (A) t-test (B, C, E, and H) one-way ANOVA (G) two-way ANOVA. Data represented as mean ± SEM.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Derivative Assay, Infection, Staining, Translocation Assay, Expressing

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 treatment abrogates T cell activation by Dendritic Cells (A) CD4 cell proliferative capacity measured in a mixed leukocyte reaction of T cells and moDC’s, with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment ( n = 3). (B) CXCR3 surface expression on CD4 T cells co-cultured with moDC’s with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment. (C and D) Cytokine secretion by moDCs or MDMs with A. fumigatus infection (MOI = 1), FK506 and IFN-γ treatment ( n = 3), (C) TNFa, (D) IL-2, (E) IL-10 and (F) IL-12. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 (A–F) one-way ANOVA. Data represented as mean ± SEM.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Activation Assay, Infection, Expressing, Cell Culture

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: IFNγ and FK506 treatment alters the transcriptomic response to A. fumigatus in both moDCs and MDMs MoDC’s and MDM’s from healthy volunteers ( n = 3) were pre-treated with FK506 and/or IFNγ, then infected with A. fumigatus (MOI = 1) for 1 h prior to sequencing (A) Gene expression heatmap of top 200 genes (ranked by SD) per unclustered condition. (B) Venn diagram showing overlap of differentially expressed genes in infected moDCs and MDMs.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection, Sequencing, Gene Expression

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: All genes differentially expressed with the FK506 treatment of A. fumigatus infected moDC’s and MDMs

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: Effects of IFNγ on moDC and MDM transcriptional responses during A. fumigatus infection (A) Numbers of differentially expressed genes with IFNγ treatment in moDC’s and MDM’s, with and without A. fumigatus infection. (B and C) Gene ontology of upregulated genes with IFNγ treatment in (B) moDCs and (C) MDMs. (D) Number of differentially expressed genes in infected cells with IFNγ treatment, with and without FK506 treatment.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: All genes differentially expressed by IFNγ treatment during the FK506 treatment of infected moDC’s and MDMs

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection

Journal: iScience

Article Title: Interferon-gamma rescues dendritic cell calcineurin-dependent responses to Aspergillus fumigatus via Stat3 to Stat1 switching

doi: 10.1016/j.isci.2024.111535

Figure Lengend Snippet: FK506 and IFNy treatment alter the epigenetic landscape of A. fumigatus infected DCs (A) Sites of differential H3K27ac binding with the A. fumigatus infection of untreated moDC’s. (B and C) Genome-wide distribution of differential H3K27ac signal around nearest gene transcription start site (TSS, green line) with the A. fumigatus infection of untreated moDCs. (D) Sites of differential H3K27ac binding with the IFN-γ treatment of A. fumigatus infected FK506-treated moDCs. (E and F) Genome-wide distribution of differential H3K27ac signal with respect to nearest gene transcription start site (TSS, green line) with the IFN-γ treatment of A. fumigatus , FK506-treated moDCs.

Article Snippet: MoDC’s at 1x10 7 per flask were pre-treated with FK506 and/or IFNγ and infected with swollen A. fumigatus conidia for 1 h. Flasks were pipetted-washed to harvest, with the aid of 10 min of cold 5 mM EDTA, and transferred to 1.5 mL Eppendorf tubes.

Techniques: Infection, Binding Assay, Genome Wide