Polyg

To explore the underlying mechanism by which exosomes regulate phagocytic processes by macrophages, the phagocytic receptor MARCO antagonist PolyG (Sigma) was applied to both in vitro cell culture and in vivo animal models.
For the SCI model, the mice were randomly divided into BMSC-Exo treatment groups and BMSC-Exos plus PolyG (2 mg/kg body weight, Sigma) treatment groups. The exosomes administered in combination with PolyG were mixed with hydrogel and then injected locally on the surface of the injured spinal cord according to our previously described method (Cao et al., 2021 (link)). For the cell culture study, macrophage cells were precultured with 200 μg/ml of PolyG (Sigma) for 30 min. Then, myelin debris uptake by the macrophages was evaluated with or without added BMSC-Exos.

Immature DCs were harvested and seeded in a 48-well plate at a concentration of 2 × 10⁶ cells/mL. Cells were supplemented with different concentrations of intact protein or peptides or polyG (Sigma-Aldrich) and incubated for 30 min at 37 °C in 5% CO₂. Then, lipopolysaccharide (LPS) (100 ng/mL), Escherichia coli Nissle 1917 (MOI 10), or Lactobacillus acidophilus NCFM (LaNCFM) (MOI 10) was added. Cytokine secretion from DCs was measured in cell culture supernatants after 20 h of incubation, using duoset ELISA (R&D systems, Minneapolis, MN, USA) for murine IL-10 (DY417), IL-12p70 (DY419), IL-23 (DY1887), and IFN-β (DY8234) according to the manufacturer’s instructions.

Total RNA was isolated from HeLa cells using the RNeasy Mini Kit (QIAGEN). mRNA was isolated from total RNA using the DynabeadsTM mRNA purification Kit (ThermoFisher). The tangled total RNA was prepared according to published work [20 (link)]. Poly(A) (Sigma-Aldrich, Cat#10108626001), Poly(C) (Sigma-Aldrich, Cat#P4903), Poly(G) (Sigma-Aldrich, Cat#P4404), Poly(U) (Sigma-Aldrich, Cat#P9528), and Ribosomal RNA (Bioworld, Cat#11020001-2) were all purchased. The 5’UTR-KpnB1-nanoLUC mRNA was transcribed with an mMESSAGE T7 kit (Invitrogen).