hoechst 33 342 staining solution  (Beyotime)

Journal: CNS Neuroscience & Therapeutics

Article Title: Forsythoside A Alleviates Neuroinflammatory Damage via Inhibiting TLR4 / NF ‐ κB / NLRP3 Activation‐Induced Astrocyte Pyroptosis in Cerebral Ischemia–Reperfusion

doi: 10.1002/cns.70867

Figure Lengend Snippet: FA suppresses OGD/R astrocyte pyroptosis and inflammation through hindering NLRP3 inflammasome activation. (A–B) Hoechst 33,342 and PI staining showed that 80 μM FA reduced pyroptosis in U251 cells after OGD/R, whereas NLRP3 agonist Nig resulted in increased pyroptosis (20×, 100 μm). (C) LDH assay showed that LDH release from U251 cells was increased following OGD/R FA inhibited LDH release, while Nig led to a rebound in LDH release. (D–E) Immunofluorescence co‐localization measured that OGD/R resulted in increased GSDMD‐N fluorescence intensity in U251 cells, FA treatment decreased GSDMD‐N level, and Nig attenuated the effect of FA (20×, 100 μm). (F–I) ELISA indicated that OGD/R resulted in increased IL‐1β, TNF‐α, IL‐18, and IL‐6 levels; FA treatment declined these inflammatory factors, but addition of Nig resulted in increased levels. (J–M) Western blot measured that OGD/R caused elevated iNOS, IL‐1β, and GSDMD‐N levels in U251 cells, and FA treatment decreased these protein levels, whereas the addition of Nig caused a rebound in these protein levels. (N) SEM to observe the morphological changes associated with pyroptosis in U251 cells. *** p < 0.001 vs. Control; ** p < 0.01 vs. OGD/R; && p < 0.01 vs. 80 μM FA.

Article Snippet: Hoechst 33,342 staining solution (C1025, Beyotime) was introduced and cultured for half an hour at 4°C.

Techniques: Activation Assay, Staining, Lactate Dehydrogenase Assay, Immunofluorescence, Fluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: LIMK2 inactivation suppresses mechanical stimulation-induced dermal fibroblast differentiation and resistance to apoptosis

doi: 10.1038/s41598-026-37610-y

Figure Lengend Snippet: ( a ) Fluorescence staining with annexin V (yellow), propidium iodide (PI) (red), and Hoechst 33,342 nuclear staining dye (blue). ( b ) The percentage of apoptotic cells (annexin V-positive, PI-negative cells, and annexin V/ PI-double-positive cells) was calculated by observing under a microscope after fluorescent staining. Mechanical stimulation reduced apoptosis in the two control groups. Baseline apoptosis was high in LIMK2-IA-mutant-GFP-HDFs, and mechanical stimulation did not reduce apoptosis. * p < 0.05. ## p < 0.01 vs control HDFs and GFP-HDFs. ( c , d ) Quadrants showing annexin V/PI flow cytometry plots: viable cells (annexin V − /PI −), early apoptotic cells (annexin V + /PI −), late apoptotic cells (annexin V + /PI +), and necrotic cells (annexin V − /PI +). The percentage of apoptotic cells was quantified using flow cytometry: (c) HDFs; (d) KDFs. LIMK2 inactivation increased apoptosis both in HDFs and KDFs. ** p < 0.01.

Article Snippet: Collected cells were washed twice with PBS and stained with Apoptosis detection reagent (GFP-Certified Apoptosis/Necrosis Detection Kit, Enzo Life Sciences, NY, USA) and Hoechst 33,342 solution (Nacalai Tesque, Kyoto, Japan).

Techniques: Fluorescence, Staining, Microscopy, Control, Mutagenesis, Flow Cytometry