cd11b fractions  (Miltenyi Biotec)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle-encapsulated microRNA-192: An anti-inflammatory adjuvant that enhances vaccine efficacy in aged mice

doi: 10.1016/j.omtn.2025.102784

Figure Lengend Snippet: F-LNP-miR-192 downregulates STAT1 and STAT3 in vivo (A, B) RAW264.7 cells were treated with mock, 5 μL of F-LNP-NC (100 pmol), or 5 μL of F-LNP-miR-192 (100 pmol) for 24 h, followed by stimulation with 200 ng/mL of LPS for 12 h. Total RNA was extracted, and STAT1 mRNA levels were measured via RT-qPCR, normalized to GAPDH levels (A). Cell lysates prepared from stimulated cells were subjected to SDS-PAGE, and proteins were detected with the indicated antibodies (B). (C) GSEA was performed for the cytokine and cytokine receptor pathway, as well as the cellular senescence pathway, with normalized enrichment scores (NES) and each p value was calculated. (D) SV (80 μL) was mixed with 20 μL of F-LNP-NC, F-LNP-miR-192, or PBS and then subcutaneously injected into young BALB/c mice. Sera were collected 24 h after injection, and serum IL-6 levels were measured using ELISA. (E) F-LNP-miR-192 (20 μL) was subcutaneously injected into young mice, and then the skin tissues were excised on day 0, 1, 2, or 3 post-injection. CD11b + and CD11b − cells were sorted using MACS beads. Total protein content in each sample was adjusted to 30 μg, subjected to SDS-PAGE, and proteins were detected via western blotting using indicated antibodies.

Article Snippet: Cells were washed with magnetic-activating cell sorting (MACS) buffer and separated into with CD11b + and CD11b − fractions using MicroBeads (Miltenyi) and MS column.

Techniques: In Vivo, Quantitative RT-PCR, SDS Page, Injection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Lipid nanoparticle-encapsulated microRNA-192: An anti-inflammatory adjuvant that enhances vaccine efficacy in aged mice

doi: 10.1016/j.omtn.2025.102784

Figure Lengend Snippet: LNP-miR-192 improves vaccine efficacy in aged mice (A and B) K-LNP-empty, K-LNP-miR-192, F-LNP-NC, or F-LNP-miR-192 or S-LNP-miR-192 (20 μL of each) was mixed with SV (80 μL) and subcutaneously injected into aged BALB/c mice ( n = 6). A second dose of the same vaccine was administered 3 weeks later. Sera were collected 4 weeks after the first-dose vaccination, and serum HI titers for influenza type A and B were determined. Data are means ± SEMs, with dots representing individual mice. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001: Log-transformed data were analyzed by Welch’s one-way ANOVA (A) and Welch’s t test (B). (C) Aged BALB/c mice were subcutaneously vaccinated once or twice with SV (80 μL) mixed with 20 μL of the indicated LNPs. Four weeks after the first vaccination, sera were collected and HI titers were measured. Data are means ± SEMs, with dots representing individual mice. ∗ p < 0.05: Kruskal-Wallis test with Dunn’s multiple comparisons test. (D) Aged BALB/c mice were subcutaneously vaccinated with SV (80 μL) mixed with F-LNP-miR-192 (20 μL). Two weeks later, draining lymph nodes were excised, and the number of GC B cells was measured via flow cytometry. Data are means ± SDs, with dots representing individual mice. ∗∗ p < 0.01; t test. (E and F) Aged BALB/c mice were subcutaneously vaccinated once or twice with SV (80 μL) alone or SV formulated with 20 μL of F-LNP-miR-192 or F-LNP-NC, as indicated. Four weeks after the first vaccination, mice were challenged with influenza A virus and monitored daily for survival. Mice losing >25% of their initial body weight were euthanized and recorded as dead. (E) Survival curves for mice vaccinated with SV alone or SV combined with F-LNP-miR-192. A significant trend in survival across all four groups was detected via the log rank test for trends ( p < 0.05). (F) Survival comparison between the SV + F-LNP-miR-192 and SV + F-LNP-NC groups, with a significant difference in survival observed (log rank test, p < 0.05). (G) SV (80 μL) was mixed with one of the indicated LNPs (20 μL) and subcutaneously injected into aged mice. Sera were collected 24 and 48 h post-injection, and serum IL-6 concentrations were measured using ELISA. Data are means ± SDs (n = 3, nonsignificant [NS] > 0.05: one-way ANOVA). (H) Aged mice were subcutaneously vaccinated using the WVP vaccine (80 μL) of influenza A virus with PBS or F-LNP-miR-192 twice at a 2-week interval. One month after the first vaccination, sera were collected, and HI and neutralization titers (NT) were determined. Data are means ± SDs. (n = 5, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001: log-transformed data were analyzed by one-way ANOVA). (I, J) 20 μL of F-LNP was subcutaneously injected into aged mice. The skin tissues were excised at 2 days after infection (I) or at indicated days (J), and CD11b + cells were isolated using MACS beads. Total protein content in each sample was adjusted to 30 μg, subjected to SDS-PAGE, and proteins were detected via western blotting using indicated antibodies.

Article Snippet: Cells were washed with magnetic-activating cell sorting (MACS) buffer and separated into with CD11b + and CD11b − fractions using MicroBeads (Miltenyi) and MS column.

Techniques: Injection, Transformation Assay, Flow Cytometry, Virus, Comparison, Enzyme-linked Immunosorbent Assay, Neutralization, Infection, Isolation, SDS Page, Western Blot