thp 1 asc gfp cell line  (InvivoGen)

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: ScRNAseq gene expression profiling of THP-1 macrophages stimulated with PyV-derived VLPs. (A) Experiment outline. (B) UMAP representation of scRNAseq data. Gray dots denote cells from all conditions combined. Black dots highlight cells from a given condition. Numbers at the bottom indicate the number of single-cell transcriptomes post-filtering. The conventional mitochondrial count filter (to remove dead cells) was intentionally omitted to enable the estimation of the fraction of dead cells. (C) Volcano plots showing bulk-like differential genes expression analysis results. Differentially expressed genes (DEGs) were defined as having an absolute fold change > 1.5 and FDR < 0.05 (Mann–Whitney U test). (D) Top: Venn diagram showing the overlap between sets of genes enriched in KIPyV VLP-treated cells relatively to control, MCPyV VLP-treated cells relatively to control, and MCPyV VLP-treated cells relatively to KIPyV VLPs. Bottom: GO enrichment analysis results for each subset of genes shown in the Venn diagram. n.s., no significant GO term enrichment. Data information: To prepare (A) , https://biorender.com/was used.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Expressing, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: ScRNAseq revealed different cell populations within THP-1 macrophages treated with PyV-derived VLPs. (A) UMAP colored by cell population annotation either combining all conditions (left) or split by condition (right). Percentages of cells per population for all data combined (B) or split by condition (C) . Results of population-enriched gene identification, including a population dendrogram (D) based on hierarchical clustering (correlation distance measure, average linkage) by enriched gene expression (E) . Selected genes are highlighted at the bottom. Results of GO enrichment analysis on selected genes groups are shown on top. CP10K—counts per 10,000. (F) Relative abundances of each population by condition. As this analysis is sensitive to the total number of cells sampled by condition, cell counts from each condition were first normalized to 10,000. (G) Relative abundance of cells at different cell cycle stages defined based on gene expression scoring.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: PyV-derived VLPs and NLPs of measles and mumps viruses were taken up by THP-1 macrophages; however, NLPs did not induce cell activation. THP-1 macrophages were treated with recombinant viral proteins (20 µg/ml) for 24 h. Nigericin (10 μM) was used as a positive control. (A) Cells were immunostained with anti-NLP and anti-VLP monoclonal antibodies (red), anti-CD68–macrophage and lysosomal marker (green), nuclear stain Hoechst33342 (blue) and analyzed by fluorescence microscopy. The negative control–secondary antibody alone is referred as a control. Images were taken using 40× objective. The scale bars indicate 100 μm. Representative images of one experiment are shown. (B) PI (dead cells) and Hoechst (all cells) nuclear staining. Images were taken using 20× objective. The scale bars indicate 200 μm. (C) Quantification of dead cells. (D) Cytotoxicity assessed by LDH assay. (E) TNF-α and (F) IL-1β secretion determined by ELISA. Data are represented using box plots, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test; stars show statistically significant results compared to control.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Activation Assay, Recombinant, Positive Control, Marker, Staining, Fluorescence, Microscopy, Negative Control, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: PyV-derived VLPs induced release of inflammatory cytokines TNF-α and IL-6 and activated NLRP3 inflammasome in human THP-1 macrophages. Macrophages were treated with PyV-derived VLPs (20 µg/ml) for 24 h. Inhibitor MCC950 (1 μM) was added 30 min before treatment. (A) TNF-α, (B) IL-6, and (C) IL-1β secretion determined by ELISA. Cytotoxicity assessed by (D) LDH assay and (E) PI and Hoechst nuclear staining. PI indicates dead cells (red) and Hoechst stains all cell nuclei (blue). Data are represented using box plots with dots showing independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test; ns, not statistically significant.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: PyV-derived VLPs induced ASC speck formation in human THP-1 macrophages. THP-1-ASC-GFP macrophages were treated for 24 h with PyV-derived VLPs (20 µg/ml); inhibitor MCC950 (1 μM) was added 30 min before treatment. (A) Formation of ASC specks was visualized by a fluorescence microscope. Representative images of one experiment are shown. Gray arrows show ASC specks. Red rectangles show magnified parts. “+” refers to MCC950 pre-treatment. The images were taken using 20× objective The scale bars indicate 200 μm. (B) Quantification of ASC speck count per cell. Data are represented using box plots with dots showing independent experiments, * p < 0.05, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test. (C, D) Time lapse of PyV VLP-induced cell activation. (C) LDH assay performed of cell culture supernatant in wild-type THP-1 macrophages. (D) The formation of ASC specks detected in THP-1-ASC-GFP. Representative images of one experiment are shown. N = 1.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Fluorescence, Microscopy, Comparison, Activation Assay, Lactate Dehydrogenase Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: PyV-derived VLPs induced caspase-1 activation in human THP-1 macrophages. Macrophages were treated for 15 h with PyV-derived VLPs (20 µg/ml); inhibitor MCC950 (1 μM) was added 30 min before treatment. (A) Cleaved caspase-1 was determined in cell supernatants by WB. The WB fragment of cleaved caspase-1 (p20 fragment) is shown. See Supplementary Figure 3 for full WB image. “+” refers to MCC950 pre-treatment. Duplicates of one experiment are shown. (B) Quantification of p20 fragment of caspase-1; duplicates of each experiments were quantified, N =2. (C) Representative images of the activated caspase-1 staining by FLICA (green) reagent. Dead cell nuclear stain PI (red) and nuclear stain Hoechst (blue) was used. The images were taken using 20× objective. The scale bars indicate 100 μm. (D) Caspase-1 quantification according to FLICA analysis, n = 40 photos per condition. N = 1. Data are represented using box plots showing number of photos and analyzed by Kruskal–Wallis test with Dunn’s post hoc , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Activation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: Cathepsin B inhibitor reduced VLP-induced IL-1β release while pan-cathepsin inhibitor decreased both IL-1β release and cell death in human THP-1 macrophages. Macrophages were treated with PyV-derived VLPs (20 µg/ml) for 24 h. Inhibitors CA-074 Me (2 or 10 μM) and K777 (15 μM) were added 30 min before treatment. (A, H) IL-1β secretion by ELISA. Cytotoxicity assessed by (B, D, F) LDH assay and (C, E, G) PI and Hoechst nuclear staining. PI indicates dead cells (red) and Hoechst stains all cell nuclei (blue). Data are represented using box plots with dots showing independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test; ns, not statistically significant.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Staining, Comparison

Journal: Frontiers in Immunology

Article Title: Activation of NLRP3 Inflammasome by Virus-Like Particles of Human Polyomaviruses in Macrophages

doi: 10.3389/fimmu.2022.831815

Figure Lengend Snippet: K + ion efflux inhibitor reduced PyV VLP-induced cell death and IL-1β release in human THP-1 macrophages. Macrophages were treated with PyV-derived VLPs (20 µg/ml) for 24 h Inhibitor glybenclamide (50 μM) was added 30 min before treatment. (A) Cytotoxicity assessed by LDH assay. (B) IL-1β secretion determined by ELISA. Data are represented using box plots with dots showing independent experiments, * p < 0.05, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: Human cell line THP-1 monocytes–ASC speck reporter cells were purchased from Invivogen (#thp-ascgfp, Invivogen, France) and called THP-1-ASC-GFP.

Techniques: Derivative Assay, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Activation of Human NK Cells by Bordetella pertussis Requires Inflammasome Activation in Macrophages

doi: 10.3389/fimmu.2019.02030

Figure Lengend Snippet: B. pertussis induces NLRP3 inflammasome activation in human MΦ-like THP-1 cells. MΦ-like THP-1 cells were stimulated with B. pertussis (Tohama I, MOI = 100, 10 or 1) or left untreated for 22 h in the presence (dashed bars) or absence (clear bars) of the caspase inhibitor, Z-VAD-FMK. (A) IL-1β ( n = 5) and (C) IL-6 ( n = 3) were measured in the supernatant of at least three independent experiments. (B) LDH release ( n = 3) was determined with a Cytotoxicity Assay. LDH release is shown as a percentage of the LDH released relative to the percentage of LDH released in the positive control, LPS + nigericin (100% cell death), for NLRP3 activation. (D–F) MΦ-like NLRP3 deficient THP-1 cells (horizontal lines) were incubated with B. pertussis (Tohama I, MOI = 10) for 22 h. (D) IL-1β ( n = 3) and ( F ) IL-6 ( n = 3) levels were measured in the supernatant using ELISAs. (E) The LDH released by MΦ-like NLRP3 deficient THP-1 cells ( n = 4) was shown as relative to the LDH release from fully lysed cultures. Results are expressed as medians with interquartile range from at least three independent experiments. Black dots represent the average values from each experiments.

Article Snippet: The THP-1, THP-1 ASC-GFP, and THP-1 NLRP3 deficient cell lines (InvivoGen) were cultured in RPMI culture medium at 37°C and 5% CO 2 .

Techniques: Activation Assay, Cytotoxicity Assay, Positive Control, Incubation