Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Validation of B16F0-Tph1 cell line. ( a ) Representative fluorescence microscopy image showing GFP expression in B16F0-Tph1 cells. ( b ) Flow cytometry analysis demonstrating 85% GFP-positive cells at passage 2 (P2). Red histogram: non-modified B16F0 cells; green histogram: B16F0-Tph1 cells. ( c ) 5-HT concentration in culture supernatants showing sustained production in B16F0-Tph1 cells across passages (P1-P5) compared to B16F0 controls. Values represent median ± IQR from n = 3 independent experiments per condition. Statistical significance was determined using Kolmogorov–Smirnov test (* p < 0.05).

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Expressing, Flow Cytometry, Modification, Concentration Assay

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Macroscopic dissemination of tumor cells over time and blood 5-HT elevation in RV following intrahepatic B16F0-Tph1 implantation. ( a ) Representative images of mice injected with 1000 B16F0-Tph1 cells in the liver, showing progression of tumor spread at 5, 6 and 7 weeks post-injection. White arrows indicate extrahepatic dissemination (peritoneal carcinomatosis at 6 weeks and mediastinal lymph node metastases at 7 weeks). No extrahepatic dissemination was detected at 5 weeks, establishing this timepoint as optimal for the main study. ( b ) Blood 5-HT concentrations measured in the right ventricle (RV) and systemic circulation 5 weeks after intrahepatic implantation in B16F0-Tph1 and B16F0 mice. Data are presented as individual points with median ± IQR; n = 3 per group. Statistical significance was determined using Kolmogorov–Smirnov test (* p < 0.05 and ** p < 0.01).

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Injection

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Analysis of urinary 5-HT and metabolite. ( a ) Log-transformed urinary serotonin (5-HT/creatinine) levels in sham (n = 7) and B16F0-Tph1 (n = 7) mice at 5 weeks post-surgery. ( b ) Log-transformed urinary 5-hydroxyindoleacetic acid (5-HIAA/creatinine) levels in the same groups. Data are shown as individual points with median ± IQR. Statistical significance was determined using Kolmogorov–Smirnov test (**** p < 0.0001, ns: not significant).

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Transformation Assay

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Heterogeneous signal intensity distribution in pulmonary and tricuspid valves of B16F0-Tph1 mice. ( a ) Representative HREM images with grayscale intensity overlays (blue: low intensity; yellow: high intensity). Sham valves show uniform low-intensity signal, while B16F0-Tph1 valves exhibit spatially heterogeneous intensity patterns, with distinct high-intensity regions adjacent to low-intensity zones. ( b ) Normalized intensity histograms demonstrate a rightward shift in B16F0-Tph1 mice, with new high-intensity voxel populations (120–180 range) absent in sham controls. Scale bar: 0.5 mm.

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques:

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: High-resolution episcopic microscopy (HREM) analysis reveals carcinoid-induced pathology in the tricuspid valve subvalvular apparatus of B16F0-Tph1 mice. Representative HREM images of the tricuspid valve (white arrow) and associated chordae tendineae (white asterisk) in Sham ( a ) and B16F0-Tph1 ( b ) mice. Quantitative analysis shows increased chordae tendineae length ( c ) and thickness ( d ) in B16F0-Tph1 compared to Sham mice. Data are shown as individual points with median ± IQR; n = 7 per group. Statistical significance was determined using Kolmogorov–Smirnov test (*** p < 0.0001, * p < 0.05, ns: not significant). Scale bar: 0.5 mm.

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Microscopy

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Cardiac valve thickness patterns. Valve thickness was measured at 6 equidistant points from free edge (position 1) to base (position 6) in B16F0-Tph1-treated mice (red lines) and sham controls (blue lines) in all cusps and leaflets (triangle = anterior, circle = posterior or left, inverted triangle = septal or right). Solid lines are spline curves of actual data (dotted lines). Data are presented as median ± IQR; n = 7 per group.

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques:

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Valvular thickness and length analysis. Comparison between B16F0-Tph1 mice (red dots) and sham controls (blue dots) across different valve leaflets and cusps. Given the absence of position effect (Fig. ), the six measurements along each leaflet/cusp were used as repeated measures. Tricuspid valve showed significant thickening across all cusps, while pulmonary valve demonstrated significant thickening in anterior and left cusps but not the right cusp. Both tricuspid and pulmonary valves showed decreased length in anterior cusps, with additional reductions in posterior (tricuspid) and left (pulmonary) cusps. Data are presented as median ± IQR; n = 7 per group. Statistical significance was determined using Kolmogorov–Smirnov test (*** p < 0.0001, ** p < 0.0001, * p < 0.0001, ns: not significant).

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Comparison

Journal: Scientific Reports

Article Title: Selective right heart valve remodelling in a mouse model of carcinoid disease revealed by high-resolution episcopic microscopy

doi: 10.1038/s41598-025-16064-8

Figure Lengend Snippet: Volumetric analysis and correlation of cardiac valves with urinary 5-HT. ( a ) 3D rendering examples of measured volumes for pulmonary and tricuspid valves in sham (blue) and B16F0-Tph1 (red) mice. ( b ) Quantitative comparison of valve volumes showing significantly increased volumes in B16F0-Tph1 mice (n = 7) compared to sham (n = 7) and B16F0 controls (n = 2) for both pulmonary and tricuspid valves. Data are presented as median ± IQR. ( c ) Linear regression analysis showing the relationship between urinary log(5-HT/creatinine) and valve volume (mm 3 ) in tricuspid valve (left) and pulmonary valve (right). Blue dots represent sham animals and red dots represent B16F0-Tph1 animals. Solid lines indicate linear regression with 95% confidence intervals (dashed lines). Dotted vertical and horizontal lines represent threshold values calculated using stepwise regression, yielding high diagnostic performance (pulmonary valve: 86% sensitivity and specificity; tricuspid valve: 100% sensitivity and specificity) for detecting pathological valve thickening. Values are presented as median ± IQR. Scale bar: 0.5 mm. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (*** p < 0.001; ns, not significant).

Article Snippet: B16F0 mouse melanoma cells from American Type Culture Collection (ATCC, USA) were genetically modified to produce 5-HT.

Techniques: Comparison, Diagnostic Assay

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Conditional expression of death inducing proteins . (A) Schematic overview of the constructs used to establish the regulatory system. The vector pWHE644 represents the regulator construct. A human EF1α promoter constitutively transcribes a tricistronic mRNA. This mRNA contains the reverse transactivator rtTA2 S -M2 (blue arrow), the transsilencer tTS D -PP (yellow arrow), and a selection marker (puromycin resistance; gray arrow). Translation of the latter two genes is mediated by internal ribosome entry sites (IRES; open boxes) from polio-virus (PV) and encephalomyocarditis- virus (EMCV). The vector pWHE655 contains the response unit used for stable transfections. It features the target gene (red arrow) driven by the Tet-responsive promoter TRE tight (open box, broken arrow) and flanked by two repeats each of a 250 bp sequence from the chicken HS4 insulator (blue triangles). A murine phosphoglycerate kinase 1 promoter (PGK; broken arrow) drives expression of a gene mediating G418-resistance. PolyA sites in all vectors are marked by a “ ⊥.” (B) Schematic representation of the cytotoxic test proteins. The residues that border the active domains expressed in the experiment are indicated above their respective closed box. A methionine added to allow translation is represented by a star. (C) Schematic overview of the regulatory system. In the OFF-State, a transsilencer (white) binds to the minimal promoter (open boxes, broken arrow) and actively suppresses transcription (cross). In the ON-State, doxycycline (blue circles) binds to both transsilencer and reverse transactivator (black). The former dissociates from, the latter binds to the minimal promoter and activates transcription (gray arrow). (D) Response of the regulatory system to different doxycycline concentrations. The B16F10-tBid transfected cell line was incubated for 24 h with various concentrations of doxycycline and mortality was measured, shown for one representative experiment out of three performed. Concentrations between 5 and 10 μg/ml showed the highest extent of cell death. An additional control at 10 μg/ml Doxy with the parental stably transfected cell line B16F10-644 was included to discard doxycycline toxicity at higher concentrations as cause of cell death (dark green diamond). Cell viability at time point “0” is shown as light green diamond.

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Construct, Plasmid Preparation, Selection, Marker, Virus, Transfection, Sequencing, Incubation, Control, Stable Transfection

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Six parameter classification by flow cytometry of the cell death phenotype of dying and dead B16F10 cells . Cell death analysis is based on morphological features (FSc and SSc), on the exposure of PS (annexin A5-FITC) and plasma membrane ion selectivity (PI), on the mitochondrial membrane potential [DiIC1(5)] and on nuclear DNA content (Hoechst 33342) detected by flow cytometry. Note: after proper gating, up to eight physiologically different subpopulations can be recorded. Dot plots exemplarily show B16F10-revCasp-3 cells after 18 h of doxycycline (5 μg/ml) treatment (A) . Rapid cell death occurred after 6 h in tBid-expressing cells and more than 95% cell death was observed after 24 h. In the presence of various caspase inhibitors [z-VAD-fmk, z-DEVD-fmk (caspase-3 inhibitor) and Ac-LEHD-cmk (caspase-9 inhibitor); all 50 μM], a significant increase in the stressed cell fraction displaying low-mitochondrial potential was observed (B) . Expression of revCasp-3 in B16F10 cells induced cell death after 24 h in more than 80% of the cells. z-VAD-fmk (50 μM) completely inhibited doxycycline-driven apoptosis. Note: stressed cells do not arise in this type of cell death induction (C) . Expression of CpnT CTD induced cell death in more than 90% of the cells after 18 h. Note: primary necrosis was the most common type of cell death observed and death occurred independently of caspase activity (50 μM z-VAD-fmk) (D) . Lethal UVB irradiation (240 mJ/cm 2 ) of parental B16F10 cells causes a rather slow progressing kind of cell death. Note: in the presence of z-VAD-fmk (50 μM), a significant increase of the stressed cell fraction displaying low-mitochondrial potential was observed (E) . Heat shock (56°C, 30 min) caused immediate necrosis in 100% of cells independent of caspase activity (F) . Displayed are the mean values from three independent experiments of relative percentages of each cell phenotype during 48 h of culture (B–F) .

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Flow Cytometry, Clinical Proteomics, Membrane, Expressing, Activity Assay, Irradiation

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Reactive oxygen species (ROS) production by dying B16F10 melanoma cells . Cells were induced to die by conditional expression of the death proteins tBid, revCasp-3, and CpnT CTD or by UVB irradiation, stained with the ROS sensor DCFH and with PI and analyzed by flow cytometry (A) . Inhibition of ROS production was performed by treatment with N -acetyl-cysteine (NAC) or mitoTEMPO, 100 μM, respectively, and recorded at 9 h after death induction (B) . Mean and SEM values of the mean fluorescence intensities of FL1 in viable cells (PI-negative) are displayed for different time points. At least three independent experiments were performed (Two and one stars indicate statistical significance at the p < 0.001 and p < 0.05 levels, respectively).

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Expressing, Irradiation, Staining, Flow Cytometry, Inhibition, Fluorescence

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Growth of B16F10 melanoma cells in the allogeneic host and concomitant immunity . Four million viable B16F10 cells (VC) were implanted s.c. in the right flank of BALB/c mice. Mice developed tumors reaching their maximum size after 2–3 weeks, followed by rejection [ (A) , black line]. Mice implanted with 4 million UVB-irradiated cells did not develop primary tumors [ (A) , purple line]. After challenge with 2 million viable cells s.c. on the left flank, those mice bearing primary tumors did not develop secondary tumors [ (B) , black line], while mice primarily inoculated with irradiated cells developed tumors similar to those of the naïve group [ (B) , purple and green lines, respectively]. Mean values ( n = 8) and the SEM are displayed. Time points showing statistical significance when compared to the group of mice implanted with VC are highlighted. Two stars and one star indicate statistical significance at the p < 0.01 and p < 0.05 levels, respectively. The two way ANOVA test corrected by Bonferroni was applied in this experiment.

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Irradiation

Journal: Frontiers in Immunology

Article Title: The Progression of Cell Death Affects the Rejection of Allogeneic Tumors in Immune-Competent Mice – Implications for Cancer Therapy

doi: 10.3389/fimmu.2014.00560

Figure Lengend Snippet: Immune response against dead or dying allogeneic tumor cells . BALB/c mice ( n = 5) were immunized in the right flank s.c. (single dose) with B16F10 dying/dead cells. After 10 days, mice were challenged s.c. in the left flank with 2 million viable cells of the parental cell line B16F10-644. Tumor growth was monitored for 30 further days (A) . Cell death was induced by UVB irradiation; heat shock; doxycycline-controlled expression of death proteins tBid, revCasp-3, and CpnT CTD . Displayed are the mean values ( n = 5) of relative tumor volumes and SEM [ (B) , * p < 0.05 after Mann–Whitney U test] and the integral of tumor size [ (C) , total tumor mass]. Inverse association between ROS production and total tumor mass developed in the allogeneic host (D) .

Article Snippet: The C57BL/6 mouse-derived melanoma cell line B16F10 bearing the haplotype H2b was purchased from ATCC (#CRL-6475) and propagated in DMEM supplemented with 10% FBS and penicillin–streptomycin (D10) at 37°C in a 5% CO 2 atmosphere.

Techniques: Irradiation, Expressing, MANN-WHITNEY

Journal: Molecular Therapy. Nucleic Acids

Article Title: Local CpG- Stat3 siRNA treatment improves antitumor effects of immune checkpoint inhibitors

doi: 10.1016/j.omtn.2024.102357

Figure Lengend Snippet: Combination treatment with intra-tumoral CpG- Stat3 siRNA and systemic PD-1 antibody reduces growth of both treated and distal tumors C57BL/6 mice were injected with 2 × 10 5 B16 melanoma cells on both right and left flanks. The left flank tumors were treated by intra-tumoral injections of CpG- Stat3 siRNA, i.p injection of anti-PD-1 antibody, or CpG- Stat3 siRNA and anti-PD-1 every other day, starting day 7 post-tumor challenge (2 × 10 5 B16-F10 cells/tumor), n = 7–8. Both treated tumor (A) and distal tumor (B) size was monitored every other day. Data are shown as means ± SEM, Student’s t test and one-way ANOVA were used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001). (C) In vivo study design for (A) and (B).

Article Snippet: Either mouse melanoma cell line B16 (ATCC) or human melanoma cell line A2058 (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1× AA.

Techniques: Injection, In Vivo

Journal: Molecular Therapy. Nucleic Acids

Article Title: Local CpG- Stat3 siRNA treatment improves antitumor effects of immune checkpoint inhibitors

doi: 10.1016/j.omtn.2024.102357

Figure Lengend Snippet: Intra-tumoral CpG- Stat3 siRNA and systemic PD-1 antibody combined treatment significantly increases CD8 + T cell tumor infiltration and activity in treated and distal tumors Single-cell suspensions from the B16 tumors from mice with indicated treatments were analyzed by flow cytometry for CD8 + (A) and CD8 + /CD4 + FoxP3 + immune cells (B) as well as IFNγ + or GzmB + CD8 + (C) cells from the indicated treatments. Data were shown as means ± SEM, n = 3 ( n is for number of samples, each of which was pooled from 2–3 mice). One-way ANOVA was used for statistical analysis (∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001).

Article Snippet: Either mouse melanoma cell line B16 (ATCC) or human melanoma cell line A2058 (ATCC) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS and 1× AA.

Techniques: Activity Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer T cell activation increases iNOS + CD206 - M1 macrophage and controls the growth of solid tumor

doi: 10.1186/s40425-019-0697-7

Figure Lengend Snippet: Intratumoral NKT cells show increased CD62L expression, and low activation marker and proliferation. B16F10 cells (1 X 10 6 cells/mouse) were s.c. injected in the naïve C57BL6 mice. a On day 5 and 13 of B16F10 injection, CD3 + NK1.1 + cells were analyzed using flow cytometry. A representative dot plot showing the NKT cell population is shown (left panel). Cells shown in the dot plots are gated on the lymphocytic gate (based on FSC-A vs. SSC-A scatter) followed by singlet populations (FSC-A vs. FSC-W scatter). Numbers in the dot plot indicate the percentage of cells. The mean percentage of NKT cells in the spleen and tumors are plotted (right panel). n = 8–10 mice/group for day 5; and n = 17 mice/group for day 13. b At day 13, NKT cells were analyzed. The dot plots showing CD69 and CD62L expression after gating on NKT cells (left). The bar represents mean, and each dot represents individual mouse (right). n = 5–8 mice/group. c B16F10 cells (1X10 6 cells/mouse) were s.c. injected in the naïve C57BL6 mice, and also intraperitoneally given BrdU (150 μg/mouse) twice a day for three constitutive days. At day 15, immune cells were stained with anti-BrdU mAb and analyzed after gating on NKT cells (left). The error bar represents s.e.m., and each dot represents data from an individual mouse (right). n = 4–5 mice/group. Student’s t -test ( a , b , c ). In all panels, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; NS, not significant

Article Snippet: Mouse melanoma B16F10 cell line was received from the national cell repository of National Centre for Cell Science, Pune, India, and maintained in complete high-glucose DMEM culture medium [DMEM with 10% FBS (Gibco), NaHCO 3 (1.5 g/liter), penicillin (50 units/ml), streptomycin (50 μg/ml) and sodium pyruvate (1 mM)] at 37 °C in a humidified 5% CO 2 incubator.

Techniques: Expressing, Activation Assay, Marker, Injection, Flow Cytometry, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer T cell activation increases iNOS + CD206 - M1 macrophage and controls the growth of solid tumor

doi: 10.1186/s40425-019-0697-7

Figure Lengend Snippet: Intratumoral NKT cells show altered expression of cytokines and cytokine receptors . Naïve C57BL6 mice were given s.c. injection of B16F10 cells (1 X 10 6 cells/mouse). a At day 13, spleen and tumors were harvested. The single cell suspension was stimulated with PMA/ionomycin, and intracellular cytokines expression was analyzed after gating on NKT cells. The representative contour plots are shown (left panel), and data from all the mice are shown (right panel). n = 3–6 mice/group. b On day 13, the surface expression of CD25 (IL-2Rα), CD122 (IL-2Rβ) and IFN-γR on NKT cells were analyzed (left). n = 4–8 mice/group. The bar represents s.e.m., and each dot represents data from an individual mouse ( a , b ). Student’s t -test ( a , b ). In all panels, * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant

Article Snippet: Mouse melanoma B16F10 cell line was received from the national cell repository of National Centre for Cell Science, Pune, India, and maintained in complete high-glucose DMEM culture medium [DMEM with 10% FBS (Gibco), NaHCO 3 (1.5 g/liter), penicillin (50 units/ml), streptomycin (50 μg/ml) and sodium pyruvate (1 mM)] at 37 °C in a humidified 5% CO 2 incubator.

Techniques: Expressing, Injection, Suspension

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer T cell activation increases iNOS + CD206 - M1 macrophage and controls the growth of solid tumor

doi: 10.1186/s40425-019-0697-7

Figure Lengend Snippet: α-GalCer increases the frequency of NKT cells, IFN-γ secretion, and inhibits tumor growth. Naïve C57BL6 mice were given s.c. injection of B16F10 cells (1 X 10 6 cells/mouse), and animals were also given i.v. injection of NK1.1 mAb (PK136; 100 μg/mouse/injection) on day − 3, + 1, + 5, + 10 and + 15 (day with respect to tumor cell injection). α-GalCer (2 μg/mouse/i.p injection) was given on day + 1, + 5, + 10, + 15 and + 20. a The tumor area was calculated and plotted. n = 6 mice/group. The data shown are representative of two independent experiments. b On day 13, spleen and tumor tissues from α-GalCer-treated mice were stained with TCR-β, α-GalCer-loaded CD1d-tetramer and nuclear stain DAPI. Images were acquired using a fluorescent microscope, and the representative images of spleen and tumor from α-GalCer treated mice are shown (magnification 200X). c On day 13, NKT cells in the spleen and tumor were analyzed using flow cytometry. Representative contour plots are shown (left panel) and mean percentages of NKT cells are plotted (middle panel). The absolute number of cells were calculated and plotted (right panel) n = 5 mice/group. d On day 13, IFN-γ, IL-4 and IL-17A expression in the NKT cells in the spleen were analyzed and plotted. n = 5–6 mice/group. e On day 13, based on CD62L and CD44 expression, memory NKT cell subsets were analyzed and plotted. n = 5–6 mice/group. f On day 23, the mean percentages of NKT cells and IFN-γ + -producing NKT cells in the spleen were analyzed. The bar represents mean, and each dot represents an individual mouse. n = 4–5 mice/group. ( b - e ). One-way ANOVA ( a ), Student’s t -test ( c - e ), * p < 0.05; ** p < 0.01; ns, not significant

Article Snippet: Mouse melanoma B16F10 cell line was received from the national cell repository of National Centre for Cell Science, Pune, India, and maintained in complete high-glucose DMEM culture medium [DMEM with 10% FBS (Gibco), NaHCO 3 (1.5 g/liter), penicillin (50 units/ml), streptomycin (50 μg/ml) and sodium pyruvate (1 mM)] at 37 °C in a humidified 5% CO 2 incubator.

Techniques: Injection, Staining, Microscopy, Flow Cytometry, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer T cell activation increases iNOS + CD206 - M1 macrophage and controls the growth of solid tumor

doi: 10.1186/s40425-019-0697-7

Figure Lengend Snippet: α-GalCer-treatment increase the frequency of IFN-γ-producing CD8 + T cells and Th1 cells. Naïve C57BL6 mice were given s.c. injection of B16F10 cells (1 X 10 6 cells/mouse) and also injected anti-NK1.1 mAb and treated with α-GalCer as in Fig. a. a At day 13, IFN-γ + production in the splenic and intratumoral CD8 + T cells was analyzed after gating on lymphocytic gate followed by singlet populations. n = 4–5 mice/group. b At day 13, CD4 + IFN-γ + T cells were analyzed after gating on CD4 + cells. n = 4–5 mice/group. The bar represents s.e.m., and each dot represents data from an individual mouse. Student’s t -test. ** p < 0.01, ns, not significant

Article Snippet: Mouse melanoma B16F10 cell line was received from the national cell repository of National Centre for Cell Science, Pune, India, and maintained in complete high-glucose DMEM culture medium [DMEM with 10% FBS (Gibco), NaHCO 3 (1.5 g/liter), penicillin (50 units/ml), streptomycin (50 μg/ml) and sodium pyruvate (1 mM)] at 37 °C in a humidified 5% CO 2 incubator.

Techniques: Injection

Journal: Journal for Immunotherapy of Cancer

Article Title: Natural killer T cell activation increases iNOS + CD206 - M1 macrophage and controls the growth of solid tumor

doi: 10.1186/s40425-019-0697-7

Figure Lengend Snippet: a-GalCer-treated mice show a higher frequency of M1 macrophages and low tumor growth. Naïve C57BL6 mice were given s.c. injection of B16F10 cells (1 X 10 6 cells/mouse) and animals were treated with α-GalCer injection (2 μg/mouse/i.p injection) on the day + 1, + 5, + 10, and + 15 (day with respect to tumor cell injection). a At day 20, the percentage of F4/80 + CD11b + cells in the spleen and tumor were analyzed. n = 3–5 mice/group. b At day 20, the percentage of iNOS + cells (M1 macrophage; left panel) and CD206 + cells (M2 macrophage; right panel) in the spleen were analyzed after gating F4/80 + CD11b + cells. n = 3–5 mouse/group. c iNOS + F4/80 + cells (M1 macrophage) in the spleen and tumor were analyzed by immunofluorescence microscopy and the representative images are shown. Original magnification 400x. d CD206 + F4/80 + cells (M2 macrophage) in the spleen and tumor of DMSO and α-GalCer-treated mice were analyzed by immunofluorescence staining (upper panel). Original magnification 400x. Representative contour plots of CD206 + F4/80 + cells (M2 macrophage) are shown (lower panel). e F4/80 + cells were depleted by i.v. injection of anti-F4/80 mAb or anti-Gr1 mAb on the day − 1, + 5, + 10 and + 15 with respect to tumor cell injection. Along with F4/80 + cell depletion, α-GalCer (2 μg/mouse/injection) was given on the day + 1, + 5, + 10, + 15 and + 20. Tumor growth was monitored, and the tumor area was calculated and plotted. n = 4–5 mice/group. f At day 20, IFN-γ expression in the splenic NKT cells were analyzed and plotted. n = 3–5 mice/group. The bar represents s.e.m., and each dot represents an individual mouse. ( a , b , f ). One-way ANOVA ( e ). Student’s t -test ( a , b , f ). * p < 0.05; ** p < 0.01; ns, not significant

Article Snippet: Mouse melanoma B16F10 cell line was received from the national cell repository of National Centre for Cell Science, Pune, India, and maintained in complete high-glucose DMEM culture medium [DMEM with 10% FBS (Gibco), NaHCO 3 (1.5 g/liter), penicillin (50 units/ml), streptomycin (50 μg/ml) and sodium pyruvate (1 mM)] at 37 °C in a humidified 5% CO 2 incubator.

Techniques: Injection, Immunofluorescence, Microscopy, Staining, Expressing