il 12 p70 levels  (R&D Systems)

Journal: International Journal of Nanomedicine

Article Title: IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8 + T, and CD4 + T Cells

doi: 10.2147/IJN.S442446

Figure Lengend Snippet: CPP transferred IL-12 into B16F10 cells and CPP-IL-12 induced immunogenic cell death. ( A ) The structure of IL-12 vector. ( B ) Western blot detection of IL-12 expression. ** P < 0.01, Student’s t -test. ( C ) ELISA. CPP-IL-12 increased IL-12 levels in the supernatant of B16-F10 cells, and 2.0 W/cm 2 of laser further increased IL-12 levels. ** P < 0.01, ANOVA. ( D ) Live/dead staining assay. Scale bars=100 μm. ( E ) The number of dead cells. CPP-IL-12 increased and 2.0 W/cm 2 of laser further increased the number of dead cells. ** P < 0.01, ANOVA. ( F ) Cell apoptosis analysis. CPP-IL-12 increased and laser treatment further increased the number of apoptotic cells. ( G ) Immunofluorescence. Green color, CALR expression. Scale bars=2 μm. ( H, I ) ELISA analysis of extracellular and intracellular HMGB1 levels, respectively. * P < 0.05, ** P < 0.01, ANOVA. (J) ATP levels. The ATP levels decreased in CPP-IL-12-treated cells, and laser treatment further reduced ATP content. ** P < 0.01, ANOVA. ( K ) CPP increased CD80 + /CD86 + expression of DC.

Article Snippet: IL-12 and HMGB1 levels were estimated using ELISA kits (CSB-E04600m and CSB-E08225M, CUSABIO, Wuhan, China) in accordance with the manufacturer′s instructions.

Techniques: Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: Journal of Nanobiotechnology

Article Title: Cu-doped TiO 2 nanoparticles improve local antitumor immune activation and optimize dendritic cell vaccine strategies

doi: 10.1186/s12951-023-01844-z

Figure Lengend Snippet: A Histogram displaying the ratio of CD8 + T cells over CD4 + T cells obtained upon in vitro stimulation by OVA-peptide loaded DCs that had either been left untreated (control) or were activated classically, or upon exposure to 33% Cu-doped TiO 2 NPs. B , C Histograms displaying the relative level (control: 100%) of B) granzyme B release, or C) perforin release from T cells obtained from part A that had been exposed to the OVA-derived SIINFEKL peptide. D , E Histograms displaying the level of D) IL12p70 release or E) relative Ccr7 surface expression (control: 100%) on DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. F , G) Histograms displaying the F) number of migrated DCs or G) the level of IL12p70 secreted from migratory DCs obtained from DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs and subsequently exposed to the CCR7 ligand 6C-kine. H , I Histograms displaying the in vivo antibody titer for H) OVA-specific IgG1 or I) OVA-specific IgG2α obtained upon intravenous administration of OVA-pulsed DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. J Histogram displaying the level of IFNγ obtained from ex vivo splenocytes obtained from animals receiving the different types of OVA-pulsed DCs and incubated with OVA ex vivo. K Tumor volumes obtained from OVA-expressing E0771 tumors grafted subcutaneously in C57Bl6 mice receiving the different OVA-pulsed DCs that had either been left untreated (control) or were activated classically, or exposed to 33% Cu-doped TiO 2 NPs. L , M Histograms displaying the relative level (control: 100%) of L) granzyme B release, or M) perforin release from CD8 + T cells isolated from OVA-E0771 tumor-bearing animals having received OVA-pulsed DC grafts of the different DC types and then exposed to the OVA-derived SIINFEKL peptide ex vivo. N Histogram displaying the level of OVA-specific CD8 + T cells isolated from the spleen of OVA-E0771 tumor-bearing animals having received OVA-pulsed DC grafts of the different DC types

Article Snippet: Media were then removed and fresh media (5 ml) was given with either no additions (controls), 33% Cu-doped TiO 2 NPs at 40 μg/ml (NP condition) or addition of maturation factors IL1β (25 ng/ml) TNFα (50 ng/ml) and IFNγ (1000 units/ml) (all from PeproTech; this is the “classical” activation scheme) for 24 h. Media were then removed and cells were incubated with soluble recombinant CD40L (BioTechne) at 16 μg/ml for 24 h after which the supernatants was collected and used to determine IL12p70 levels by ELISA (M1270, R&D Systems).

Techniques: In Vitro, Control, Derivative Assay, Expressing, In Vivo, Ex Vivo, Incubation, Isolation

Journal: Cancer immunology, immunotherapy : CII

Article Title: Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

doi: 10.1007/s00262-012-1248-y

Figure Lengend Snippet: Fig. 2 VRP-IL-12 infection of DC leads to IL-12 secretion and maturation of DC. Murine den- dritic cells were generated from bone marrow of mice by cultur- ing cells in the presence of murine GM-CSF and IL-4 for 10 days. DCs were infected with VRP-IL-12 at MOI 10 or MOI 100, or empty vector (VRP- Empty) at MOI 100, and incu- bated for 18–24 h. Lipopolysac- charide (1 g/mL)-induced IL-12 secretion served as a posi- tive control. a mIL-12 p70 ELISA was performed on cul- ture supernatants.*p < 0.001, **p < 0.0001 compared to mock DC. b VRP-IL-12-infected DCs (MOI 100) or LPS-treated DCs were incubated with Brefeldin A for 4 h, Wxed/permeabilized and stained with anti-mouse IL-12 antibody. Percentages of IL-12- positive populations are shown in each dot plot

Article Snippet: Culture supernatants from VRP-IL-12-infected DC or VRP-Empty-infected DC were analyzed for IL-12 p70 level using Quantikine mouse IL-12 p70 ELISA kit (R&D Systems, Minneapolis, MN).

Techniques: Infection, Generated, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay, Incubation, Staining

Journal: PLoS ONE

Article Title: Endoplasmic Reticulum Aminopeptidase-1 Functions Regulate Key Aspects of the Innate Immune Response

doi: 10.1371/journal.pone.0069539

Figure Lengend Snippet: CD11c+ dendritic cells were isolated from C57BL/6 WT (n = 4) or ERAP1-KO mice (n = 6) and in vitro stimulated with specified concentrations of rEA protein. Cultured media was used to perform an IL12p70 ELISA as described in Materials and Methods. The bars represent Mean ± SEM. 0.1 ng/ml of rEA is an optimal stimulation dose. Statistical analysis was completed using two-tailed homoscedastic Student’s t-tests; *, - indicate values, statistically different between WT_rEA and ERAP1-KO_rEA groups, p<0.05. Representative of three independent experiments is shown.

Article Snippet: Following incubation, culture medium was analyzed for mouse IL12p70 levels using an ELISA kit and following its enclosed instructions (R&D Systems, Minneapolis, MN).

Techniques: Isolation, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test