Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Characterization of Res-PD-L1@nmEVs . (A) Schematic illustration of the Res-PD-L1@nmEVs synthesis procedure. (B-D) Representative transmission electron microscopy (TEM) images, dynamic light scattering (DLS) size distributions, and zeta potential measurements of nEVs, PD-L1@mEVs, PD-L1@nmEVs, and Res-PD-L1@nmEVs. (E) PD-L1 expression in PD-L1-overexpressing MSCs (OE-PD-L1) and negative control (NC) MSCs, and CD11b expression in HL60 cells before and after DMSO stimulation, as determined by Western blot. (F) Expression levels of neutrophil membrane markers (CD11b, CXCR2, RAGE, TLR2) and the exosomal marker CD63 in the four EV types. (G) Fluorescence co-localization images of DiO-labeled nEVs (green) and DiL-labeled PD-L1@mEVs (red) after fusion, demonstrating hybrid vesicle formation. (H) Size stability of Res-PD-L1@nmEVs stored at 4 °C and 37 °C for 7 days. (I-K) Binding and neutralization capacity of Res-PD-L1@nmEVs against inflammatory cytokines (TNF-α, IL-6, IL-1β) in vitro. ∗ vs. 0ug/ml; # vs. 100 μg/ml, p < 0.05, n = 5.

Article Snippet: For detecting neutrophil characteristic proteins, the primary antibodies comprised CD11b (HA722075, HUABIO, China), CXCR2 (ER1906-87, HUABIO, China), RAGE (ET1702-27, HUABIO, China), TLR2 (ET1705-92, HUABIO, China), TNFR1(RT1624, HUABIO, China), IL6R ( AWA43078 , Abiowell, China) and the exosome marker CD63 (ET1607-2, HUABIO, China).

Techniques: Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Expressing, Negative Control, Western Blot, Membrane, Marker, Fluorescence, Labeling, Binding Assay, Neutralization, In Vitro

Journal: Bioactive Materials

Article Title: Inhalable PD-L1-engineered hybrid cellular vesicles suppress excessive neutrophil activation and restore mitochondrial homeostasis to alleviate ischemia–reperfusion lung injury and pneumonia

doi: 10.1016/j.bioactmat.2026.03.024

Figure Lengend Snippet: Res-PD-L1@nmEVs Suppresses Neutrophil Activation and Preserves Mitochondrial Integrity via PD-L1 Delivery (A-B) Rats subjected to lung IRI received nebulized administration of different formulations (Res, nEVs, PD-L1@mEVs, PD-L1@nmEVs, or Res-PD-L1@nmEVs) before ischemia and after reperfusion. Lung tissues were collected 2 h post-reperfusion. (A) Representative immunofluorescence images showing the expression and localization of CD11b (green), MPO (red), and PD-1 (yellow) in lung sections across treatment groups. (B) Enlarged view of the IRI group from (A). (C-D) mRNA levels of CD95 (C) and CD206 (D) in lung tissues. (E-F) Levels of myeloperoxidase (MPO) (E) and matrix metalloproteinase-9 (MMP-9) (F) in bronchoalveolar lavage fluid (BALF). (G-I) (G) Representative transmission electron microscopy (TEM) images of lung tissues (scale bar: 2 μm). (H) Proportion of damaged mitochondria. (I) Average number of mitophagic events per cell. (J) Immunofluorescence co-localization of mitochondrial marker TOMM20 (red) and EpCAM (green) in lung tissues (nuclei stained with DAPI, scale bar: 50 μm). (K-L) Protein expression levels of Beclin-1 (K) and LC3 (L) in lung tissues, with insets showing immunofluorescence co-localization of Beclin-1 (green) and LC3 (red) across treatment groups (nuclei stained with DAPI, scale bar: 50 μm). ∗ vs. Sham; # vs. IRI; & vs. IRI + PD-L1@nmEVs, p < 0.05.

Article Snippet: For detecting neutrophil characteristic proteins, the primary antibodies comprised CD11b (HA722075, HUABIO, China), CXCR2 (ER1906-87, HUABIO, China), RAGE (ET1702-27, HUABIO, China), TLR2 (ET1705-92, HUABIO, China), TNFR1(RT1624, HUABIO, China), IL6R ( AWA43078 , Abiowell, China) and the exosome marker CD63 (ET1607-2, HUABIO, China).

Techniques: Activation Assay, Immunofluorescence, Expressing, Transmission Assay, Electron Microscopy, Marker, Staining

Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Representative workflow of isolation and culture of adult mouse microglia (A and B) Dissect the brain into small pieces on ice in Petri dish. (C) Collect cell pellets in C-tubes following mechanical/enzymatic dissociation using a gentleMACS dissociator. (D) Preparation of cell straining and debris removal processes. (E) Perform debris removal by carefully overlaying ice-cold PBS onto the cell suspension. (F) After centrifugation, identify three layers; the middle, yellowish layer corresponds to myelin dna debris. (G) Enrich CD11b+ cells by magnetic separation using appropriate columns. (H) Seed isolated microglia onto 6-wll culture plates for downstream assays.

Article Snippet: CD11b Microbead (Clone: M1/70.15.11.5) (1:10 ratio) , Miltenyi Biotech , Cat#130-049-601; RRID: AB_2927911.

Techniques: Isolation, Suspension, Centrifugation

Journal: STAR Protocols

Article Title: Protocol for isolating and culturing microglia from the adult mouse brain using a magnetic-activated cell sorting system

doi: 10.1016/j.xpro.2026.104471

Figure Lengend Snippet: Flow cytometry for MACS-isolated microglia purity (A and B) Total CD11b-positive cells from MACS columns; (C) Cell viability assessed by Zombie Red staining; (D) Infiltrating leukocytes, including neutrophils (Ly6G+) and T lymphocytes (CD3+); (E) Proportions of microglia (CD11b+CD45low) versus monocytes/border-associated macrophages (CD11b+CD45high); (F) Proportion of resting (homeostatic) microglia identified as CD11b+TMEM119+ cells.

Article Snippet: CD11b Microbead (Clone: M1/70.15.11.5) (1:10 ratio) , Miltenyi Biotech , Cat#130-049-601; RRID: AB_2927911.

Techniques: Flow Cytometry, Isolation, Staining