mab anti mouse il 1β  (Bio X Cell)

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of siRNA cytotoxicity and silencing efficiency. ( A ) XTT assay results indicating cell viability ( left ) and LDH assay results measuring cytotoxicity ( right ) in THP-1 Mφ treated with 40 nM of siRNA candidates (siIL-1β.1, siIL-1β.2, siIL-1β.3) or controls (transfection medium Opti-MEM, transfection reagent RNAiMAX, and siCon). ( B ) Schematic of the psiCHECK™-2 dual-luciferase reporter plasmid. Renilla luciferase (Rluc) serves as the primary reporter, with siRNA target sequences (TS) cloned downstream of the reporter gene. Firefly luciferase (Fluc) is co-expressed for normalization. ( C ) Relative Ren/Luc activity in HeLa cells co-transfected with siRNAs at 20 nM ( left ) or 40 nM ( right ). Relative luciferase activity was normalized to siCon set as 100%. Data are represented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as *** p ≤ 0.001, **** p ≤ 0.0001, and ns (non-significant, p > 0.05). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: XTT Assay, Lactate Dehydrogenase Assay, Transfection, Luciferase, Plasmid Preparation, Clone Assay, Activity Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: IL-1β protein knockdown in differentiated THP-1 cells post-siRNA transfection. Cells were treated with 100 ng/mL PMA for 72 h to induce differentiation into THP-1 Mφ. Following differentiation, cells were stabilized in PMA-free medium for 24 h before transfection with 20 nM siRNA. At 43 h post-transfection, cells were stimulated with 100 ng/mL LPS for 5 h to induce an immune response. Non-stimulated, non-transfected THP-1 Mφ served as the control condition. ( A ) Representative Western blot showing pro-IL-1β protein expression under various treatment conditions, including the addition (+) or absence (−) of PMA, LPS, and siRNA transfection. β-actin served as a loading control to confirm equal protein loading (20 μg per lane). ( B ) Densitometric quantification of pro-IL-1β protein levels from Western blots, normalized to β-actin and expressed relative to siCon set as 100%. ( C ) Relative IL-1β protein levels quantified by ELISA, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: Knockdown, Transfection, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of cell viability and nebulization setup for siRNA delivery. ( A ) Custom nebulizer chamber for siRNA delivery, incorporating the PARI SINUS2 nebulizer with 3D-printed adapters for a 6-well plate. The setup includes an aspirator outlet to prevent mist leakage. ( B ) XTT assay results for THP-1 Mφ: ( Top ) Viability after removal of medium for varying time points up to 30 min. ( Bottom ) Viability after nebulization with Opti-MEM for up to 15 min. ( C ) Live-dead staining of THP-1 Mφ: ( Top ) Control cells cultured without nebulization. ( Bottom ) Cells nebulized with 20 nM siRNA (siIL-1β.2) for 5 min. Live cells are indicated in green and dead cells in red. Data are represented as mean ± SD from three independent experiments (n = 3), ns indicates a non-significant difference ( p > 0.05). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test. Scale bars = 200 μm.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: XTT Assay, Staining, Control, Cell Culture, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Evaluation of siRNA delivery and functionality post-nebulization. ( A ) Fluorescence microscopy of THP-1 Mφ transfected with fluorescent-labeled siCon.Cy3 via nebulization. Shown are DAPI nuclear staining (blue), phase contrast, siCon.Cy3 signal (red), and the merged overlay. ( B ) Dual-luciferase reporter assay after sequential transfection, in which THP-1 Mφ were first transfected with the reporter plasmid and subsequently with siRNA 6 h later. Relative luciferase activity was normalized to siCon set as 100%. ( C ) Cell viability assessment by XTT assay in HeLa cells following temporary medium removal for up to 30 min. ( D ) Dual-luciferase reporter assay following siRNA delivery via nebulization, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001 and ns, non-significant ( p > 0.05). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons or unpaired Student’s t -test. Scale bars = 20 μm.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: Fluorescence, Microscopy, Transfection, Labeling, Staining, Luciferase, Reporter Assay, Plasmid Preparation, Activity Assay, XTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Functional siRNA Delivery via Jet Nebulization: Proof-of-Concept IL-1ß Silencing in Macrophage-like THP-1 Cells

doi: 10.3390/ijms27062915

Figure Lengend Snippet: Validation of nebulized siRNA for endogenous IL-1β knockdown in differentiated THP-1 cells. Cells were treated with 100 ng/mL PMA for 72 h to induce differentiation into THP-1 Mφ. Following differentiation, cells were cultured in PMA-free medium for 24 h before transfection with 20 nM siRNA using the PARI SINUS2 Nebulizer System. At 43 h post-transfection, cells were stimulated with 100 ng/mL LPS for 5 h to induce an immune response. Non-stimulated, non-transfected THP-1 Mφ served as the control condition. ( A ) Representative Western blot showing pro-IL-1β protein expression under various treatment conditions, including PMA-induced differentiation, LPS stimulation, and siRNA nebulization. The presence or absence of the respective treatment is indicated as + and −. β-actin served as a loading control to confirm equal protein loading (20 μg per lane). ( B ) Densitometric quantification of pro-IL-1β protein levels from Western blots, normalized to β-actin and expressed relative to siCon (set to 100%). ( C ) Relative IL-1β protein levels quantified by ELISA, normalized to siCon set as 100%. Data are presented as mean ± SD from three independent experiments (n = 3), with statistical significance denoted as **** p ≤ 0.0001. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison test.

Article Snippet: The membrane was incubated overnight at 4 °C with a primary antibody against IL-1β (12242, 1:1000, Cell Signaling Technologies, Danvers, MA, USA) diluted in blocking buffer.

Techniques: Biomarker Discovery, Knockdown, Cell Culture, Transfection, Control, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Comparison