Journal: Frontiers in Immunology

Article Title: Development and validation of a potency assay matrix for optimized and consistent manufacture of clinical mesenchymal stem/stromal cells

doi: 10.3389/fimmu.2026.1725191

Figure Lengend Snippet: MSCs produce high levels of EVs that drive M2 polarization. (A) Heatmap showing the log 2 fold-change in expression of genes included in the “phagocytosis” pathway between Ctrl, MSC-CM, M1, and M2 macrophages. (B) Flow cytometry data showing the expression levels of MerTK on monocytes differentiated to M1 macrophages, M2 macrophages, co-cultured with MSCs, cultured with 50% MSC-CM, or left untreated for 3 days. Data are means ± SD. n = 4 independent replicates using four different PBMC donors. (C) Representative images of monocytes cultured with EVs derived from CBO-labeled MSCs for 24hrs. (D) Time course of CBO-labeled EV uptake and expression of CD163, MerTK, CD80, and CD14 on monocytes. Data are means ± SD. n = 2 technical replicates using one PBMC donors. (E) CD163 and MerTK expression on monocytes treated with EVs isolated from MSC-CM (EVs MSC-CM) or cRPMI (EVs RPMI) for 3 days. Data are means ± SD. n = 4 independent EV preparation from one MSC donor (donor 063), using four different PBMC donors. (F) IL-10 levels in the supernatant of monocytes treated with EV isolated from MSC-CM or cRPMI for 3 days. Data are means ± SD. n = 4 independent EV preparation from one MSC donor (donor 063) and using four different PBMC donors. (G) CD163 and MerTK expression and IL-10 levels at different concentrations of EVs. (H) Percentage of CBO + monocytes of monocytes incubated with isolated CBO labeled EVs ± a MerTK inhibitor or αMerTK antibody or incubated with the EV isolation column flowthrough (FT). Data are means ± SD. n = 2 independent experiments using two different PBMC donors. (I) M-CSF ELISA levels of EV FT or EVs from MSCs. Data are means ± SD. n = 4 independent experiments using four different PBMC donors. (J) CD163 MFI of monocytes incubated with EVs isolated from cRPMI, MSC-EVs (Ctrl) or MSC-EVs plus an αM-CSF antibody, CSF1Ri, an αMerTK antibody, or MerTKi. Data are means ± SD. n = 4 independent experiments using four different PBMC donors. Statistical significance was determined using one-way ANOVA with Tukey’s HSD test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: Freshly thawed MSCs were stained with CBO (Biotium, 30022) for 20mins at 37 °C in PBS by adding 5μL of CBO per mL PBS at a cell density of 10 6 cells/mL.

Techniques: Expressing, Flow Cytometry, Cell Culture, Derivative Assay, Labeling, Isolation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: EET-Based Therapeutics Mitigate Sorafenib-Associated Glomerular Cell Damage

doi: 10.3390/biom15091324

Figure Lengend Snippet: 8,9-EET Analog Mitigates Sorafenib-Induced HRMCs Death by Suppressing Caspase 3/7 Activity. Caspase 3/7 activity, a marker of sorafenib-induced apoptosis, is visualized as green, fluorescent spots. An increase in green fluorescence indicates elevated caspase 3/7 activity and greater cell death, while a decrease reflects reduced apoptotic activity. HRMCs were seeded into 96-well plates for compound screening. Each plate was used to test two to three compounds, alongside matched vehicle and sorafenib (10 µM) controls. Caspase 3/7 activation was assessed using a luminescent assay, with control datasets applied uniformly across all compounds tested on the same plate. MDB-32, MDB-52a, and MDB-52b were each tested on separate 96-well plates, with individual sets of vehicle and sorafenib controls specific to each compound. In contrast, MDB-77 and MDB-78 were tested concurrently on a single 96-well plate, sharing a common set of vehicle and sorafenib controls. This design enabled consistent intraplate comparisons while maintaining compound-specific control conditions. ( a ) Treatment with the 8,9-EET analog MDB-32 led to a 20–40% reduction in sorafenib-induced caspase 3/7 activity, suggesting a protective effect against apoptosis. ( b ) Cells treated with MDB-52a in combination with 10 µM sorafenib showed significantly lower caspase 3/7 activity compared to sorafenib alone. MDB-52a reduced activity in a dose-dependent manner by 60–90%, demonstrating strong anti-apoptotic efficacy in HRMC cells. ( c ) A similar reduction in caspase 3/7 activity was observed with MDB-52b, further supporting its protective role. ( d , e ) HRMC cells treated with MDB-77 and MDB-78 also exhibited decreased caspase 3/7 activity relative to sorafenib-treated controls, indicating potential anti-apoptotic effects of these compounds.

Article Snippet: Caspase 3/7 activation in human renal mesangial cells (HRMCs) and podocytes was assessed using the IncuCyte ® SX5 live-cell analysis system (Sartorius, Göttingen, Germa-ny) over 48 h. Cells were seeded at a density of 5000 cells per well in 96-well TPP plates to facilitate optimal adherence.

Techniques: Activity Assay, Marker, Fluorescence, Activation Assay, Luminescence Assay, Control

Journal: Biomolecules

Article Title: EET-Based Therapeutics Mitigate Sorafenib-Associated Glomerular Cell Damage

doi: 10.3390/biom15091324

Figure Lengend Snippet: 8,9 EET Analog Mitigates Sorafenib-Induced Cell Death of Podocyte by Suppressing Caspase 3/7 Activity. The protective potential of 8,9-EET analogs against sorafenib-induced apoptosis in podo-cytes was evaluated by measuring caspase 3/7 activity, a key marker of programmed cell death. Apoptotic activity was visualized as green, fluorescent spots, an increased number of spots indi-cates elevated caspase 3/7 activity, while fewer spots suggest reduced apoptosis. Human podo-cytes were seeded into 96-well plates to evaluate caspase 3/7 activation in response to various test compounds. Each plate was configured to include two to three compounds, along with matched vehicle and sorafenib (10 µM) controls. A single set of control data (vehicle and sorafenib) was used for all compounds tested on the same plate to ensure consistent intra-plate comparisons. MDB-52a, MDB-52b, and RM-84 were tested concurrently on one 96-well plate, sharing a common set of vehicle and sorafenib controls. Similarly, MDB-77 and MDB-78 were tested together on a separate plate, also using a single set of vehicle and sorafenib controls for both compounds. Treatment with 8,9-EET analogs significantly reduced caspase 3/7 activity induced by sorafenib, indicating their protective effects: ( a ) MDB-52a: Co-treatment with MDB-52a and 10 µM sorafenib resulted in a marked reduction in caspase 3/7 activity compared to sorafenib alone. The effect was dose-dependent, with a reduction of approximately 50–70%. ( b ) MDB-52b: Similar protective effects were observed, with caspase 3/7 activity reduced by 40–60% in a dose-dependent manner. ( c , d ) MDB-77 and MDB-78: Both compounds decreased caspase 3/7 activity when combined with sorafenib. MDB-77 was effective at 1 and 3 µM, while MDB-78 showed minimal effect at lower doses but was effective at 10 µM. ( e ) RM-84: This analog also demonstrated a dose-dependent reduction in caspase 3/7 activity, with the 10 µM dose significantly lowering apoptosis compared to sorafenib-only treatment.

Article Snippet: Caspase 3/7 activation in human renal mesangial cells (HRMCs) and podocytes was assessed using the IncuCyte ® SX5 live-cell analysis system (Sartorius, Göttingen, Germa-ny) over 48 h. Cells were seeded at a density of 5000 cells per well in 96-well TPP plates to facilitate optimal adherence.

Techniques: Activity Assay, Marker, Activation Assay, Control

Journal: Advanced Science

Article Title: Endothelium‐Derived Engineered Extracellular Vesicles Protect the Pulmonary Endothelial Barrier in Acute Lung Injury

doi: 10.1002/advs.202306156

Figure Lengend Snippet: Uptake and distribution of LET‐EVs and EVs in vivo and in vitro. A) Fluorescence microscope of cellular uptake of DIL‐labeled LET‐EVs and EVs after 2 and 24 h of incubation with HPMVECs. The stains used were as follows: DIL‐labeled EVs (Orange), F‐actin (Green), and DAPI (Blue) (Scale bar: 25 µm). B) Quantification of EVs integrated fluorescence density based on ImageJ analysis. The uptake efficiency of DIL‐labeled LET‐EVs was higher than that of DIL‐EVs. C) Imaging of ALI mice after 0, 2, and 48 h of administration of DIR, DIR‐EVs, and DIR‐LET‐EVs. Compared with the DIR and DIR‐EVs groups, the fluorescence signal of LET‐EVs was mainly accumulated in the epigastric region after 2 h. D) Fluorescence imaging of tissues from ALI mice in the DIR, DIR‐EVs, and DIR‐LET‐EVs groups after 48 h. Compared with the DIR and DIR‐EVs groups, the signal in the DIR‐LET‐EVs groups was mainly aggregated in lung tissues. E) Quantitative analysis of mean fluorescence intensities in different tissues following administration of EVs and LET‐EVs. F) The clearance of EVs in lung tissues was calculated by dividing average lung signals by average liver signals. G) Uptake of DIL‐EVs and DIL‐LET‐EVs by endothelial cells in lung tissues. Immunofluorescence staining was performed in lung sections using an antibody against CD31 (endothelial cell marker, green). Nuclei were stained with DAPI (Scale bar: 100 µm). All the data are presented as the mean ± SD ( n = 3). * p < 0.05, *** p < 0.001 compared with the EVs group by unpaired Student's t ‐tests.

Article Snippet: Purified EVs were incubated with 10 μ m of the cell membrane dye DIL (Solarbio, Beijing, China) for 30 min. Unbound DIL dye was removed with three washes with PBS buffer (pH 7.4, 3000 g, 20 min per wash) in a 10 kDa MWCO Amicon centrifugal filter at 4 °C.

Techniques: In Vivo, In Vitro, Fluorescence, Microscopy, Labeling, Incubation, Imaging, Immunofluorescence, Staining, Marker