Journal: BioMed Research International

Article Title: The Comparative Utility of Viromer RED and Lipofectamine for Transient Gene Introduction into Glial Cells

doi: 10.1155/2015/458624

Figure Lengend Snippet: (a) Viromer RED shows differential viability in cell lines and primary cells: HEK293, primary rat astrocytes, primary rat Schwann cells, and BV2 microglial cell were treated with transfection reagents with or without cargo (blue = untreated control, green = Lipofectamine 3000 with enhancer, gray = Viromer RED, purple = Lipofectamine 3000 with enhancer with pGL4.13 luciferase plasmid, orange = Viromer RED with pGL4.13 luciferase plasmid, and red = 1% hydrogen peroxide (H 2 O 2 ) treated cells for 5 min before the addition of CellTiter-Fluor reagent). Cells were assessed for viability 24 hr after transfection using CellTiter-Fluor reagent. Absolute fluorescence (RFU measured at 380 nm Excitation/505 nm Emission) was normalized to mean fluorescence of nontransfected cells and fold change reported. Comparisons were made within the untreated control ( ∗ ), within the transfection reagents (with or without cargo), (&) and across the transfection reagents (within cargo or no cargo group) (#) in the respective cells/cell lines. Values represent mean ± 1 SD. ∗ , &, # P < 0.05; ∗∗ , &&, ## P < 0.01; ∗∗∗ , &&&, ### P < 0.001. (b) Viromer RED transfected cells show a higher luminescence as compared to those transfected with Lipofectamine 3000: HEK293, primary rat astrocytes, primary rat Schwann cells, and BV2 microglial cell lines were treated with transfection reagents, with or without cargo (blue = untreated control, green = Lipofectamine 3000 with enhancer, gray = Viromer RED, purple = Lipofectamine 3000 with enhancer with pGL4.13 luciferase plasmid, and orange = Viromer RED with pGL4.13 luciferase plasmid). Cells were lysed 24 hr after transfection following the assessment of viability (absolute fluorescence (RFU)) using CellTiter-Fluor reagent. Luminescence (RLU) was measured using the luciferase assay reagent. The RLU/RFU ratio data was normalized to the mean RLU/RFU ratio of the nontransfected cells. Comparisons were made across the transfection reagents (with cargo group) (#) in the respective cells/cell lines. Values represent mean ± 1 SD. ### P ≤ 0.001.

Article Snippet: The pMaxGFP plasmid vector for control transfections was obtained from Lonza (Allendale, NJ) and pGL4.13 (E668A) was procured from Promega (Madison, WI).

Techniques: Transfection, Luciferase, Plasmid Preparation, Fluorescence

Journal: Journal of Diabetes and Metabolic Disorders

Article Title: TLR4/MyD88 -mediated CCL2 production by lipopolysaccharide (endotoxin): Implications for metabolic inflammation

doi: 10.1007/s40200-018-0341-y

Figure Lengend Snippet: Inhibition of TLR4 down-regulates the LPS induced CCL2. Antibody-treated cells were stimulated with LPS and incubated for 24 h. Cells and culture media were collected. Real time PCR data showed that neutralization of TLR4 significantly suppress LPS induction of CCL2 (A) and reduced CCL2 protein was determined in the culture media by ELISA (B). TLR4 siRNA transfected THP-1 cells showed reduced expression of TLR4 mRNA compared to the cells transfected with control siRNA (C). LPS induced CCL2 expression was significantly inhibited in TLR4 deficient cells at both mRNA (D) and protein (E) levels

Article Snippet: THP-1 cells were transfected separately with TLR4-siRNA (30 nM; OriGene Technologies, Inc. MD, USA), scramble (control) siRNA (30 nM; OriGene Technologies, Inc. MD, USA, USA) and pmaxGFP (0.5μg; Amaxa Noclecfector Kit V for THP-1, Lonza).

Techniques: Inhibition, Incubation, Real-time Polymerase Chain Reaction, Neutralization, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Control

Journal: Scientific Reports

Article Title: TNFα induces matrix metalloproteinase-9 expression in monocytic cells through ACSL1/JNK/ERK/NF-kB signaling pathways

doi: 10.1038/s41598-023-41514-6

Figure Lengend Snippet: ACSL1 siRNA transfection reduced TNFα-mediated MMP-9 production. We transfected THP-1 monocytic cells with siRNA targeting human ACSL1 gene expression or scrambled siRNA (a control siRNA). ( A , B ) After 36 h, we performed real-time PCR to measure ACSL1 gene expression or western blotting for protein to test the knocking down efficiency. ( C ) We then incubated ACSL1-deficient cells with TNFα for 24 h. We determined mRNA expression of MMP-9 by real-time PCR. ( D ) We determined MMP-9 protein in culture media using ELISA. ( E ) The effect of siRNA transfection in combination with TNFα on cell viability was evaluated by measuring cell metabolic activity (MTT assay). The cell viability is expressed as the percentage of cells compared to the condition of vehicle control. Three independent experiments were performed with similar results. All data are expressed as mean ± SEM (n ≥ 3). t test or onene way ANOVA (Dunnett’s Test) for comparing treatments vs control) were used). ** p < 0.01, *** p < 0.001.

Article Snippet: We transfected the cells separately with siRNA against ACSL1 (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), scramble siRNA (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), and pmaxGFP (0.5 ug; Amaxa Nucleofector Kit V for THP-1cells, Lonza, Cologne, Germany).

Techniques: Transfection, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Activity Assay, MTT Assay