Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: Cytotoxicity of ultravist in P815 cells. P815 cells were incubated with different doses of ultravist for 24 h and cell viability was measured by the CCK-8 test. * P<0.05 vs. control.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques: Incubation, CCK-8 Assay, Control

Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: Mast cells could be activated by ultravist stimulation. P815 cells were stimulated with ultravist for 24 h; (A) histamine and (B) β-hexosaminidase were measured by ELISA. * P<0.05, ** P<0.01.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: miRNA expression profile of ultravist-stimulated mast cells. P815 cells were treated with 50 mg/ml ultravist for 24 h. miRNA from the control and ultravist treatment groups was extracted and analyzed by miRNA array. miRNA/miR, microRNA.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques: Expressing, Control

Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: KEGG Pathway Analysis of ultravist-treated mast cells. P815 cells were stimulated with 50 mg/ml ultravist for 24 h. Subsequent KEGG pathway analysis of the mTOR signaling pathway revealed upregulation of genes related to (A) miR-19A-3p and (B) miR-362-3p, which are highlighted in red. KEGG, Kyoto Encyclopedia of Genes and Genomes; miRNA/miR, microRNA.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques:

Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: miR-19a-3p and miR-362-3p could be upregulated by ultravist treatment. P815 cells were treated with different doses of ultravist and (A) miR-19a-3p and (B) miR-362-3p were detected by reverse transcription-quantitative PCR. * P<0.05, ** P<0.01. miRNA/miR, microRNA.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Biomedical Reports

Article Title: Radiocontrast medium induces histamine release in association with upregulation of miR‑19a‑3p and miR‑362‑3p expression

doi: 10.3892/br.2024.1780

Figure Lengend Snippet: Network analysis of miRNA-target interactions in mast cells. P815 cells were stimulated with 50 mg/ml ultravist for 24 h, the predicted interactions between miR-19a-3p and miR-362-3p were analyzed and the key target genes-Aox1, Akap2, and Uba2l were highlighted in yellow. miRNA/miR, microRNA.

Article Snippet: The mouse mast cell cell line, P815 (Elabscience Biotechnology, Inc.), was cultured in RPMI1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Cytiva) and 1% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37 ̊C incubator with 5% CO 2 .

Techniques:

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma

doi: 10.1007/s00262-019-02427-4

Figure Lengend Snippet: Expression of PD-1/PD-L1 on HCC tumour tissues and tumour cell lines. a Representative images of immunohistochemistry sections in the tumour tissue of HCC patient #20. Positive cells are stained brown by immunoperoxidase. Arrows mark PD-1-positive mononuclear cells (upper left), CD8+ T cells (upper right) and perforin-positive cells (lower right), respectively. PD-L1 staining shown in the lower-left panel indicates a greater number of positive mononuclear cells (macrophages and CD8+ T cells). Of note, HCC tumour cells did not stain positive for PD-1 and PD-L1. Tu tumour. b The flow cytometric analysis of PD-L1/L2 expression on P815 mouse mastocytoma cells in contrast to various hepatoma cell lines (HepG2, HepT1, Huh4, Hep3B, Huh7). Unlike hepatoma cells, which up-regulated PD-L1 and PD-L2, P815 cells remained PD-L1 and PD-L2 negative in cell culture. Thus, we used P815 cells as target cells in lectin-dependent cellular cytotoxicity (LDCC) assays to study the functional role of Tregs on T cell functions in a model of PD-L1/L2-negative tumour cells

Article Snippet: Cell line authentication of murine P815 cells was performed by the Swiss DNA company Microsynth (Supplementary Table 3).

Techniques: Expressing, Immunohistochemistry, Staining, Cell Culture, Functional Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma

doi: 10.1007/s00262-019-02427-4

Figure Lengend Snippet: IFN-gamma production and degranulation by CD8+ T cells in lectin-dependent cellular cytotoxicity (LDCC) assays and effects of checkpoint inhibition. a The gating strategy to analyse IFN-gamma production and T cell degranulation in CD8+ T effector cells by flow cytometry in co-cultures with Con A-loaded P815 cells and Tregs (Treg to Teff ratio: 1:2). b IFN-gamma production (left) and CD107a degranulation (right) of CD8+ T effector cells in LDCC assays before (BL), with exposure to Con A-loaded P815 target cells (stim = peak secretion) and upon adding Tregs (stim + Tregs). Adding autologous Tregs reduced P815-induced peak IFN-gamma secretion and degranulation of CD8+ T cells in all study groups. p values refer to significances obtained by paired Student t test marked by bars. c Provides the summary statistics concerning the differences of IFN-gamma-production (left) and CD107a degranulation (right) by CD8+ T cells after sequential addition of Con A-loaded P815 target cells and autologous Tregs (Treg to Teff ratio: 1:2), as well as in the presence of added neutralizing anti-PD-1, anti-PD-L1, and anti-CTLA-4, and anti-GITR (10 µg/ml each). Columns represent the mean ± SD of 6–18 different donors. Anti-PD-1 and anti-PD-L1—but not anti-CTLA-4 nor anti-GITR—partially reversed Treg-associated inhibition of IFN-gamma secretion in CD8+ T cells from patients with HCC. Treg-mediated inhibition of T cell degranulation was not reversed by any of the antibodies. p values refer to significances obtained by paired Student t test marked by bars

Article Snippet: Cell line authentication of murine P815 cells was performed by the Swiss DNA company Microsynth (Supplementary Table 3).

Techniques: Inhibition, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: NLRP3 expression was increased by estrogen stimulation via ER-α in MCs. (A) Bar plot of differentially expressed genes (DEGs) from the KEGG pathway enrichment analysis; a comparison between β-estradiol treated P815 cells and the control. (B) A volcano plot was used for visualization of the differentially expressed genes. The gray point in the plot represents genes with no statistical differences (-1< log 2 (FC) < 1, P > 0.05), the red point represents upregulated genes (log 2 (FC) > 1, P < 0.05), and the blue point represents downregulated genes (log 2 (FC) < -1, P < 0.05) with statistical significance. (C) Quantitative PCR analysis of ESR1 in P815 cells after ESR1-specific shRNA transfection. (D) Western blot analysis of ER-α in P815 cell nuclei after stable transfection of ESR1-specific shRNA. (E) Relative quantification of NLRP3 mRNA level in P815 was measured by FPKM reads using RNA-seq. (F) Quantitative PCR analysis NLRP3 mRNA levels in P815 cells treated with β-estradiol after knockdown of ESR1 compared with the negative control. (G) Representative images of immunofluorescence staining for NLRP3 (red) in mast cell line P815 incubated with or without 100 pmol/mL β-estradiol for 24 h Data in (C, D, F) represent three independent experiments. Data are expressed as mean ± SEM. Statistics: Unpaired Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, statistically not significant.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Transfection, Western Blot, Stable Transfection, RNA Sequencing Assay, Negative Control, Immunofluorescence, Staining, Incubation

Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: ERE sites in the NLRP3 promoter mediated estrogen-induced NLRP3 transcription. (A) ChIP analysis was performed using anti-ER-α or anti-Histone H3 antibody to ascertain the existence of the ERE in the promoter of the NLRP3 gene. The PCR results showed that a 159-bp fragment containing the presumed ERE could be precipitated after P815 cells were treated with β-estradiol for 24 h. (B) The pulled-down band was excised from the gel and sequenced. (C) Schematic diagram of luciferase reporter constructs. Basic-Luc: pGL4-basic plasmid; NLRP3-Luc: pGL4-basic plasmid with the NLRP3 promoter fragment including presumed ERE-like sequence; delNLRP3-Luc: pGL4-basic plasmid with the mutant NLRP3 promoter fragment, with the presumed ERE-like sequence deleted. (D) Luciferase activities of three report systems with or without ESR1 plasmid co-transfection in 293T cells were compared with each other. Renilla luciferase plasmid was used to normalize transfection efficiencies. The experiments were repeated three times and data are presented as means ± SEM. Statistics: Unpaired Student’s t-test. * P < 0.05, ** P < 0.01.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Luciferase, Construct, Plasmid Preparation, Sequencing, Mutagenesis, Cotransfection, Transfection

Journal: Frontiers in Immunology

Article Title: NLRP3 Inflammasome Activation of Mast Cells by Estrogen via the Nuclear-Initiated Signaling Pathway Contributes to the Development of Endometriosis

doi: 10.3389/fimmu.2021.749979

Figure Lengend Snippet: The NLRP3 inflammasome pathway was activated by estrogen via ER-α. (A) Western blot analysis of NLRP3, cleaved caspase-1, caspase-1 precursor, cleaved IL-1β, IL-1β precursor, and ASC in P815 cells treated with 100 pmol/mL β-estradiol for 1 h, 3 h, 6 h, 12 h, and 24 h. (B) Quantified results of western blot assays. (C) Intracellular concentration of K + was determined by the ratio of the fluorescence intensities obtained by exciting PBFI at 340/380 nm wavelengths while monitoring emission at 500 nm, and presented as percent of untreated control. (D) The concentration of IL-1β in the P815 cell culture supernatant was tested using ELISA. Data in (B–D) represent three independent experiments. Data are expressed as mean ± SEM. Statistics: ANOVA followed by Dunnett t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse mast cell line P815 from ScienCell Research Laboratories (CA, USA) was cultured with RPMI-1640 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (PS) (Thermo Fisher).

Techniques: Western Blot, Concentration Assay, Fluorescence, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: CD40L expressed from the canarypox vector, ALVAC, can boost immunogenicity of HIV-1 canarypox vaccine in mice and enhance the in-vitro expansion of viral specific CD8 + T cell memory responses from HIV-1-infected and HIV-1-uninfected individuals

doi: 10.1016/j.vaccine.2008.05.018

Figure Lengend Snippet: CD40L expressed by recombinant ALVAC virus boosts the immunogenicity of an HIV-1 canarypox vaccine in mice. Female Balb/c mice of 6–8 wks age were immunized 3 times with an HIV-1 canarypox vaccine, vCP1452, with either of vCPmCD40L or vCPmSP-D-CD40L or ALVAC II. Six weeks after the last immunization, mice were sacrificed and spleens were taken and used for preparation of splenocytes. (A) IFN-γ ELISpot. Splenocytes were stimulated with P815 cells pulsed with an H-2Kd restricted HIV-1 Gag peptide, AMQMLKETI, for 16 hrs. Cells producing IFN-γ were detected and counted as described in Materials and Methods. (B) and (C) MHC-I tetramer analysis. Splenocytes were stained with anti-mouse CD8 mAb and AMQMLKETI/H-2Kd tetramer. (B) shows representative flow cytometry results for each group of mice. Numbers are percentage of tetramer positive cells in CD8+ cells. (C) shows summary of the tetramer data for each group of mice. (D) to (G) Polyfunctional CD8+ T cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse IFN-γ and anti-mouse TNF-α mAbs and co-expressing cells are measured in (D) and (E) or anti-mouse IFN-γ and anti-mouse CD107a mAbs and co-expressing cells are measured in (F) and (G). (D) and (F) show representative flow cytometry results and (E) and (G) show summary of the polyfunctional CD8+ T cell data for each group of mice. (H) CD8+ T cells producing IFN-γ analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8 and anti-mouse IFN-γ mAbs. (I) and (J) Memory HIV-1-Gag specific CD8+ cells analyzed by flow cytometry. Splenocytes were stained with anti-mouse CD8, anti-mouse CD127 (IL-7Rα) mAbs, and AMQMLKETI/H-2Kd tetramer. In (I) CD8+/Tetramer+ cells are gated and measured by CD127 (y-axis). CD8+ T cells from naïve mice unstained for CD127 was used as control for CD127 gating. (J) shows summary data of CD127 expression in tetramer+ cells. (K) Lymphocyte proliferation. Splenocytes were stimulated with HIV-1 p24 Gag for 6 days and [3H]-thymidine incorporation was measured. (L) Cytokine production. Splenocytes were stimulated with HIV-1 p24 Gag for 5 days. IFN-γ and IL-4 in the supernatant were measured by ELISA. (M) Serum anti-Gag antibody. HIV-1 p24 Gag was used as antigen, and mice serum anti-Gag antibodies were measured by ELISA. Data shown are mean±SEM. n=4–10. *: P<0.05. **: P<0.01.

Article Snippet: The plates were then washed 3 times with PBS and blocked with R-10 medium (RPMI-1640 supplemented with 10% fetal bovine serum) for at least 2 h. Splenocytes and P815 cells (mouse mastocytoma cell line) that had been pulsed with or without H-2K d -resticted immunodominant HIV-1 Gag 199–207 peptide AMQMLKETI (Mimotopes Pty Ltd., Clayton Victoria, Australia) were added to the plates and incubated at 37°C for 18 h. The plates were then washed, labeled with biotinylated rat anti-mouse IFN-γ antibody (Cat No. 554410; BD Biosciences), and incubated at room temperature for 2 h with shaking.

Techniques: Recombinant, Virus, Immunopeptidomics, Enzyme-linked Immunospot, Staining, Flow Cytometry, Expressing, Control, Enzyme-linked Immunosorbent Assay