Cell Cycle and Apoptosis Analysis Kit

Protocol tips

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	- Gambogic acid is added to cells at varying concentrations (0.0, 0.5, 1.0, and 2.0 μM) for 24 h. - Cells were fixed by 75% ethanol. Incubated with RNaseA at 37 °C in a water bath for 40 min and PI solution for 20 min in dark

Protocol tips
- Gambogic acid is added to cells at varying concentrations (0.0, 0.5, 1.0, and 2.0 μM) for 24 h. - Cells were fixed by 75% ethanol. Incubated with RNaseA at 37 °C in a water bath for 40 min and PI solution for 20 min in dark

Publication protocol

Gambogic acid (98% purity, Yuanye Biotech, China) was dissolved in dimethyl sulfoxide (DMSO) to 20 mg/mL and stored for subsequent use. Gemcitabine (Sigma-Aldrich, USA) was dissolved in water to 50 mM and stored. The extracellular signal-regulated kinase (ERK) inhibitor ulixertinib, proteasome inhibitor MG-132, and pan-caspase inhibitor Z-VAD-FMK (Selleck, USA) were dissolved in DMSO to 10 mM and stored for subsequent use. All regents were stored at a temperature below − 80 °C. A solution of 4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) was dissolved in phosphate buffered saline (PBS) and stored at 20 °C. The AnnexinV/PI apoptosis kit was purchased from Vazyme (China), and a Cell cycle detection kit was purchased from Beyotime (China). Primary antibodies against cleaved PARP, cleaved caspase-3, cleaved caspase-9, Bcl-2, Bax, ERK1/2, phospho-ERK1/2, AKT, and phospho-AKT were purchased from Cell Signaling Technology (USA). Primary antibodies against RRM1, RRM2, and E2F1 were purchased from Affinity Bioscience (USA). An IHC (immunohistochemistry) detection kit was purchased from CWBio (China).


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