ampk  (Proteintech)

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: STAR Protocols

Article Title: Protocol for differentiation and efficient AAV-mediated gene delivery to hiPSC-derived microglia for functional studies

doi: 10.1016/j.xpro.2026.104455

Figure Lengend Snippet: Propagation of calcium signal within microglia after ATP stimulation (A) Baseline GCaMP8s expression. (B) Regions of interest (ROIs): one somatic (ROI 1) and two distal regions (ROI 2 and 3) were chosen. (C) Snapshots showing propagation of the fluorescence signal within the cell following ATP stimulation. (D) Normalized fluorescence traces (ΔF/F0, F0 = mean fluorescence intensity over 10 s prior to stimuli) recorded from ROIs in B. The period shaded in green indicates when ATP was present in the recording chamber. ROI 3 (most distal) exhibits spontaneous activity prior to stimulation, indicated by asterisks. Dashed vertical lines indicate time points (t0-t3) corresponding to images in C.

Article Snippet: Dulbecco’s phosphate buffered saline without Ca 2+ and Mg 2+ , DPBS (−/−) , Thermo Fisher Scientific , 14190–086.

Techniques: Expressing, Fluorescence, Activity Assay

Journal: STAR Protocols

Article Title: Protocol for differentiation and efficient AAV-mediated gene delivery to hiPSC-derived microglia for functional studies

doi: 10.1016/j.xpro.2026.104455

Figure Lengend Snippet:

Article Snippet: Dulbecco’s phosphate buffered saline without Ca 2+ and Mg 2+ , DPBS (−/−) , Thermo Fisher Scientific , 14190–086.

Techniques: Virus, Recombinant, Saline, Plasmid Preparation, Expressing, Software, Hood, Sterility, Electron Microscopy, Inverted Microscopy, Flow Cytometry, Microscopy, Cell Culture, Fluorescence, Imaging, Dispersion