mouse mast cell line p815  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: Blockade of the immunosuppressive KIR2DL5/PVR pathway elicits potent human NK cell–mediated antitumor immunity

doi: 10.1172/JCI163620

Figure Lengend Snippet: ( A – C ) Redirected cytotoxicity of KIR2DL5 + primary NK cells against P815. ( A ) The lysis of P815 cells ( n = 4). ( B ) The degranulation (CD107a) and cytokine production (IFN-γ and TNF-γ) of KIR2DL5 + primary NK cells ( n = 4). CD56 served as negative control. ( C ) Cytokine production in the coculture supernatant of KIR2DL5 + primary NK cells with the indicated antibody-coated P815 ( n = 5). Data are represented as mean ± SEM. ( D ) Lysis of scrambled control or PVR KO A427 or Jurkat cells by KIR2DL5-transduced primary NK cells (KIR2DL5/NK) or KIR2DL5 – control NK cells (Control NK) at indicated E/T ratios. ( E ) PVR-KIR2DL5–mediated inhibitory synapse formation. Left: Representative imaging of cell conjugates acquired upon sorted KIR2DL5 + primary NK contact with control-YFP/Raji (top) or PVR-YFP/Raji (bottom), followed by staining with anti-KIR2DL5 mAbs and phalloidin. Scale bars: 10 μm. Right: Intensity quantification of F-actin, YFP, and KIR2DL5 at the immunological synapses (IS) and the cell surface away from synapses (Non-IS) from KIR2DL5 + NK cell–Control Raji ( n = 25) and KIR2DL5 + NK-PVR/Raji ( n = 35) conjugates. ( F ) Lysis of scrambled control or PVR KO A427 (top) or Jurkat (bottom) cells by sorted KIR2DL5 + primary NK cells in the presence of F8B30 or mIgG1 at indicated E/T ratios. In D and F , data are mean for duplicate measurements and representative of 3 independent experiments with 3 different donors. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by 1-way ANOVA ( A and B ), 2-tailed paired Student’s t test ( C and E ), or multiple unpaired t test ( D and F ).

Article Snippet: Mouse cell lines used in this study were mouse fibroblast line NIH 3T3 (ATCC, CRL-1658), mouse mast cell line P815 (ATCC, TIB-64), and mouse myeloma cell line NSO (a gift from Matthew D. Scharff, Department of Cell Biology, Albert Einstein College of Medicine).

Techniques: Lysis, Negative Control, Control, Imaging, Staining

Journal: Cell reports

Article Title: The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections

doi: 10.1016/j.celrep.2019.12.014

Figure Lengend Snippet: (A) Representative histograms showing the expression of CD107a on CD8CD38 low and CD8CD38 high T cells from healthy subjects by flow cytometry. To measure CD107a expression, cells were stimulated with plate-coated CD3 and CD28 antibodies with Golgiplug and anti-CD107a for 5 h and stained with proper surface markers. (B) Degranulation (%CD107a) of CD8 T cells from healthy subjects and patients with SLE evaluated by flow cytometry (healthy subjects = 8, SLE = 12; Welch’s test). (C) Correlation between percentage of CD38 high and degranulation in CD8 T cells (healthy subjects = 8, SLE = 12; closed circles, healthy subjects; opened circles, SLE; Pearson’s correlation). (D–F) Degranulation (%CD107a) of CD8 T cells sorted on CD38 low and CD38 high of both healthy subjects and patients with SLE in both groups (D) and separately in healthy subjects (E) and SLE (F) by flow cytometry (healthy subjects = 8, SLE = 12; closed circles, control; opened circles, SLE; healthy subjects = 8, SLE = 12; Welch’s test). (G) Degranulation (%CD107a) of the different subpopulations of CD8 T cells sorted on CD38 low and CD38 high by flow cytometry (n = 5 healthy donors; one-way ANOVA with multiple comparison). (H) Representative dot blots (left) and cumulative data (right) showing percentage of of Annexin V + P815 cells after coincubation with CD8CD38 low or CD8CD38 high T cells from healthy subjects analyzed by flow cytometry (normal T cells = 4; Kolmogorov-Smirnov test). In all figures, average data are represented as mean ± SD.

Article Snippet: Jurkat human CD4 T cell line and P815 mouse mast cell line were obtained from ATCC were maintained in RPMI1640 (Thermo Fisher Scientific) with L-glutamine, 1% of Penicillin/Streptomycin and 10% of FBS.

Techniques: Expressing, Flow Cytometry, Staining, Control, Comparison

Journal: Cell reports

Article Title: The CD38/NAD/SIRTUIN1/EZH2 Axis Mitigates Cytotoxic CD8 T Cell Function and Identifies Patients with SLE Prone to Infections

doi: 10.1016/j.celrep.2019.12.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Jurkat human CD4 T cell line and P815 mouse mast cell line were obtained from ATCC were maintained in RPMI1640 (Thermo Fisher Scientific) with L-glutamine, 1% of Penicillin/Streptomycin and 10% of FBS.

Techniques: Control, Purification, Recombinant, Quantitation Assay, Negative Control, Plasmid Preparation, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: Prostate cancer cells-derived PKD2/3 promote chemotactic migration of mast cells and endothelial cells tube formation. a Conditional medium (CM) from PC-3 M or DU145 cells transiently transfected with si-CTL, si-PKD2, si-PKD3 was used to be chemoattractant for P815 mast cell migration by transwell assays. ***p < 0.001, **p < 0.01 versus si-CTL by one-way ANOVA . b Silencing of PKD2 and PKD3 verification by Western blotting (left panel). c VEGF mRNA level was assessed by qPCR in P815 mast cells induced by conditional medium from DU145 cells transfected with siRNA of PKD2 or /and PKD3 (right panel). *p < 0.05, **p < 0.01 versus si-CTL by one-way ANOVA . d Knockdown efficiency were verified by Western blot in PC-3 M(up) or P815 mast cells(bottom). Cells were transiently transfected with siRNA of indicated genes using lipofectmine3000. e HUVEC cells were treated with conditional medium from co-cultured of P815 mast cells transfected with si-CTL or si-VEGF and PC-3 M cells transfected with si-CTL, si-PKD2, si-PKD3 or si-PKD2 + 3. Tube formation of HUVEC cells were visualized by phase contrast inverted microscope (100×). f Tube formation was assessed the number of nodes per image was quantified and analyzed by two way ANOVA, **p < 0.01

Article Snippet: The prostate cancer cell lines, PC-3, DU145 and RM1, and the mouse mast cell lines P815 were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Derivative Assay, Migration, Transfection, Western Blot, Knockdown, Cell Culture, Inverted Microscopy

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Protein kinase Ds promote tumor angiogenesis through mast cell recruitment and expression of angiogenic factors in prostate cancer microenvironment

doi: 10.1186/s13046-019-1118-y

Figure Lengend Snippet: PKD2/3 enhance MCs migration through upregulation of SCF, CCL5 and CCL11 in prostate cancer cellsmRNA and the protein level of the scf , ccl5 and ccl11 were analyzed by real-time qPCR and ELISA in DU145 ( a ) and PC-3 M ( b ) cells transfected with siRNA of PKD2, PKD3. Data representing the means ± S.D. of three independent experiments was analyzed by one-way ANOVA for significance versus si-CTL. ***p < 0.001, **p < 0.01, *p < 0.05 versus si-CTL. c Knockdown efficiency of PKD2 and PKD3 in prostate cancer cells was verified by Western blotting. d DU145 cells were transfected with siRNA of PKD2, PKD3, the Conditional medium (CM) was collected to measure migration of P815 cells in response to SCF, CCL5, and CCL11 treatment by transwell assay. e Quantification were analyzed from data in D. ***p < 0.001 versus si-CTL by One-ANOVA tests

Article Snippet: The prostate cancer cell lines, PC-3, DU145 and RM1, and the mouse mast cell lines P815 were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Transfection, Knockdown, Western Blot, Transwell Assay