porcine inf γ elispot assay  (Mabtech Inc)

Journal: Molecular Metabolism

Article Title: Leukocyte-type 12/15-lipoxygenase is essential for timely inflammation-resolution and effective tissue regeneration following skeletal muscle injury

doi: 10.1016/j.molmet.2025.102224

Figure Lengend Snippet: Bone marrow derived-macrophages obtained from Alox15 −/− mice display shifts in secreted lipid mediator profile and impaired M2 polarization. Bone-marrow-derived macrophages (MФ) (BMMs) obtained from wild type (WT) and 12/15-LOX deficient ( Alox15 −/− ) mice were maintained in serum free media as naïve M0 MФ, or induced to differentiate into either M1 MФ by exposure to lipopolysaccharide (LPS) (100 ng/mL) and interferon gamma (IFN − γ ) (20 ng/mL), or M2 MФ by exposure to interleukin-4 (IL-4) (20 ng/mL). Following 24 h of polarization mRNA expression levels of pro-inflammatory cytokines including interleukin-1β ( Il1b ) ( A ) and interleukin-6 ( Il6 ) ( B ), or M2 MФ markers including CD206 ( Mrc1 ) ( C ) and annexin A1 ( Anxa1 ) ( D ) were measured by real-time quantitative reverse transcription PCR (RT-qPCR). E-F: BMMs from WT mice ( E ) and Alox15 −/− mice ( F ) were polarized to a M1 or M2 activation state for 48 h, fixed in 4% paraformaldehyde (PFA), and stained with primary antibodies against CD163 (green) and CD206 (red). Nuclei were counterstained by DAPI (blue). Scale bars are 200 μm. G-K: Gene expression of major lipid mediator biosynthesis enzymes including COX-1 ( Ptgs1 ) ( G ), COX-2 ( Ptsg2 ) ( H ), 5-LOX ( Alox5 ) ( I ), platelet-type 12-LOX ( Alox12 ) ( J ), and 15-LOX-1 ( Alox15 ) ( K ) in M0, M1, and M2 MФ obtained from WT and Alox15 −/− mice following 24 h of polarization. L: A heatmap of the top 30 most differentially modulated lipid metabolites detected in conditioned cell culture media samples obtained from WT and Alox15 −/− M0, M1, and M2 MФ following 24 h incubation in serum free DMEM as assessed by targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). M-R: Concentrations of major representative lipid mediator metabolites of the cyclooxygenase (COX) pathway (e.g., PGE 2 ) ( M ), 5-lipoxygenase (5-LOX) pathway (e.g., 5-HETE) ( N ), 12-lipoxygenase (12-LOX) pathway (e.g., 12-HEPE) ( O ), 15-lipoxygenase (15-LOX) pathway (e.g., 15-HETE) ( P ), cytochrome P450 (CYP 450) pathway [e.g., (9(10)-EpOME)] ( Q ), and downstream bioactive SPMs (e.g., LXA 4 ) ( R ). Bars show the mean ± SEM of cells obtained from 5 mice per group (biological replicates) with dots representing BMMs from each individual mouse. P- values were determined by two-way ANOVA followed by Holm-Šídák post-hoc tests. ∗ p < 0.05 vs. M0, & p < 0.05 vs. M1, and # p < 0.05 for WT vs. Alox15 −/− cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: MФ were allowed to adhere to the plastic surface for 24 h. M-CSF was then withdrawn and BMMs were either maintained as naïve M0 MФ in serum free DMEM, polarized to M1 MФ by stimulation with 100 ng/mL LPS (Sigma–Aldrich, L2630) and 20 ng/mL interferon gamma (INF-γ) (R&D Systems, 485-MI), or polarized to M2 MФ by stimulation with 20 ng/mL interleukin-4 (IL-4) (R&D Systems, 404-ML).

Techniques: Derivative Assay, Expressing, Reverse Transcription, Quantitative RT-PCR, Activation Assay, Staining, Gene Expression, Cell Culture, Incubation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of Virology

Article Title: E2-based mRNA vaccine encapsulated in lipid nanoparticles protects pigs against classical swine fever virus

doi: 10.1128/jvi.00978-25

Figure Lengend Snippet: Effect of pseudouridine modification on E2tm mRNA vaccine. ( A ) Particle size and polymer dispersity index (PDI) graph of Ψ E2tm mRNA-LNP. The particle sizes and PDI were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZPS (Malvern). ( B ) Expression of E2tm and Ψ E2tm mRNA-LNPs in vitro . HEK293T cells were incubated with mRNA-containing LNPs for 24 h and analyzed using western blotting. ( C ) The schematic representation of pig immunization. Pigs were immunized at days 0 and 21 with E2tm (60 or 150 µg) or Ψ E2tm (60 or 150 µg) mRNA. ( D ) Antibody levels induced by E2tm and Ψ E2tm mRNA in pigs. Serum samples were collected at 7, 14, 21, 28, 35, 42, 63, 84, and 108 dpi. The levels of CSFV-specific antibodies in serum samples were determined by blocking ELISA. ( E ) Serum neutralizing antibodies against CSFV Shimen strain over times. The titers were expressed as the highest serum dilution that protected more than 50% of the cells from infection. ( F ) The serum neutralizing antibodies against different CSFV strains, including Shimen strain, HuB100 strain, and HeBHH1/95 strain. ( G ) IFN-γ-secreting peripheral blood mononuclear cells (PBMCs) induced by E2tm and Ψ E2tm mRNA vaccines in pigs detected by ELISpot. PBMCs were isolated from serum samples collected at 28 dpi. The dot line indicates the lower limit of detection. The data were analyzed using the one-way ANOVA and presented as the mean ± SD; * P < 0.05.

Article Snippet: Porcine INF-γ ELISpot assay was conducted on PBMCs, following the manufacturer’s instructions (MabTech).

Techniques: Modification, Polymer, Expressing, In Vitro, Incubation, Western Blot, Blocking Assay, Enzyme-linked Immunosorbent Assay, Infection, Vaccines, Enzyme-linked Immunospot, Isolation