incucyte nuclight rapid red dye  (Sartorius AG)

Journal: Cells

Article Title: Extracellular Vesicles Derived from VEGF mRNA-Engineered Mesenchymal Stem Cells Promote Endothelial Cell Survival

doi: 10.3390/cells15080717

Figure Lengend Snippet: Angiogenesis induced by VEGF secreted from VEGF-MSCs. Human umbilical vein endothelial cells labelled by CytoLight Green (GFP-HUVECs) were used for the development of angiogenic networks, and the length of networks was determined by the IncuCyte live-cell analysis system. ( A ) VEGF effectively induced the formation of vascular networks, which was able to be inhibited by suramin, a VEGF signaling inhibitor. Data shown as mean (SD) ( n = 6). ( B ) A concentration-dependent effect was shown in the angiogenesis assay using CCM-VEGF with different VEGF concentrations (1, 2, 4, and 8 ng/mL). Blank control, without any VEGF supplement added; suramin group, 4 ng/mL VEGF and 100 µM suramin added. Data shown as mean (SD) ( n = 4); ns, not significant; **** p < 0.05.

Article Snippet: The following day, cells were treated with 25 nM STS in HUVEC growth medium for 24 h. To visualize apoptotic cells, IncuCyte Nuclight Rapid Red Dye (Sartorius, catalog# 4717) and IncuCyte Caspase-3/7 Green Dye (Sartorius, catalog# 4440) were added into the wells at a 1:1000 dilution.

Techniques: Cell Analysis, Concentration Assay, Angiogenesis Assay, Control

Journal: Cells

Article Title: Extracellular Vesicles Derived from VEGF mRNA-Engineered Mesenchymal Stem Cells Promote Endothelial Cell Survival

doi: 10.3390/cells15080717

Figure Lengend Snippet: VEGF-MSC-EVs exhibit superior anti-apoptotic effects on HUVECs. ( A ) Internalization of EGFP-MSC-EVs by HUVECs. EGFP-MSCs derived EVs (EGFP-MSC-EVs) labeled with ExoGlow-Protein (Red) were incubated with HUVECs pretreated with 25 nM staurosporine (STS). Real-time 3D imaging was performed using the Tomocube HT-X1 for 14 h. Blue and purple arrows indicate two distinct EV clusters. Scale bar: 10 µm. Apoptosis in HUVECs was induced by 25 nM staurosporine (STS), and apoptotic cells were visualized by colocalization of Nuclight Rapid Red Dye and Caspase-3/7 Green Dye. ( B ) MSC-EVs protected HUVECs from apoptosis in a dose-dependent manner. ( C ) Quantification of apoptotic cells 30 h post-EV treatment. The protective effects of EVs were observed at 50%, 20%, and 10% EV concentrations, while 5% EVs and equivalent volumes of vehicle controls (50%, 20%, 10%, and 5% 25 mM Trehalose) did not promote HUVEC survival. Blank control, without adding STS. Positive control, with 100 ng/mL commercial VEGF added. ( D ) VEGF-MSC-EVs demonstrated superior anti-apoptotic effects compared to EVs derived from control MSCs. ( E ) Quantification of apoptotic cells 30 h post-treatment with VEGF-MSC-EVs. An ordinary one-way ANOVA followed by Sidak’s multiple comparisons test was used for statistical analysis. Data shown as mean (SD) ( n = 4); ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001.

Article Snippet: The following day, cells were treated with 25 nM STS in HUVEC growth medium for 24 h. To visualize apoptotic cells, IncuCyte Nuclight Rapid Red Dye (Sartorius, catalog# 4717) and IncuCyte Caspase-3/7 Green Dye (Sartorius, catalog# 4440) were added into the wells at a 1:1000 dilution.

Techniques: Derivative Assay, Labeling, Incubation, 3D Imaging, Control, Positive Control

Journal: Nature Communications

Article Title: Timosaponin AIII enhances CAR-T cell potency and prevents relapse through impairing CAR-Tregs

doi: 10.1038/s41467-026-70867-5

Figure Lengend Snippet: a CD19 CAR-T cells were generated from human T cells as described in the Methods. After expansion, CAR-T cells were treated with DMSO or TAIII for 72 h. The proportion of FoxP3 + Treg in CD4 + T was then analyzed by flow cytometry. Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. b – c CAR-T and Untransduced T cells (UNT) cells were treated with TAIII (0, 0.5, 1, 2 µM) for 24 and 72 h under non-stimulatory conditions (without tumor cells). T cell proliferation and cell viability/apoptosis were assessed using CellTiter-Glo assays and flow cytometry (PI/Annexin V/7-AAD staining). Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t-test or ordinary one-way ANOVA with Dunnett’s test. CAR-T cells were co-cultured with Raji-Luc ( d ) or Nalm6-Luc ( e ) cells at effector-to-target (E:T) ratios of 1:2 or 1:4 under indicated treatments. Cytotoxicity was measured by residual luciferase activity, and IFN-γ, IL-2, and TNF-α levels were determined by ELISA. UNT cells were included as negative controls; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. f Real-time cytotoxicity of CAR-T cells co-cultured with Raji cells was monitored over 36 h using Incucyte® cytolight red and annexin V staining; Data are presented as mean ± SD ( n = 3 biological replicates); two-way ANOVA with Šídák’s multiple comparisons test. g IFN-γ and TNF-α protein levels in CAR-T–Raji co-cultures after 16 h were measured by ELISA; Data are presented as mean ± SD ( n = 3 biological replicates); two-sided unpaired t -test. h FoxP3a (FoxP3) mRNA levels in CAR-T–Raji co-cultures were determined by qRT-PCR. Data are presented as mean ± SD from independent experiments ( n = 3–4 biological replicates); two-sided unpaired t -test.

Article Snippet: For this assay, Raji cells were stained with Incucyte® cytolight rapid red dye (Sartorius, #4706) prior to seeding at a density of 10,000 cells per well in a 96-well flat-bottom plate.

Techniques: Generated, Flow Cytometry, Staining, Cell Culture, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR