oridonin compound was purchased from Beijing OKA Biological Technology Co., Ltd. (Beijing, China), and the purity was confirmed to be ≥98%. The oridonin solution was prepared in DMSO (Sigma, MO, USA). Gemcitabine hydrochloride was purchased from Selleck Chemical (Houston, TX, USA) and made up in sterile phosphate-buffered saline (PBS) with the stock solution of 160 mg/kg.
Seed cells on 96-well plates by 5000 cells/well and treat with oridonin, with, as indicated, final concentrations for a different duration. Fix cells in situ with 40% (w/v) cold Trichloroacetic acid (TCA). Let them sit for 5 min. Incubate plates at 4 °C for 1 h. Discard supernatant and gently wash plates 5 times with water, followed by an air-dry. Stain fixed cells with 0.4% (w/v) SRB with 1% acetic acid as solubilizer. After staining, wash with 1% acetic acid to remove the free dye and air-dry. Solubilize the bound stain with 100 mM Tris Base for reading. Read absorbance on a microplate reader at 515 nm [45 (link),46 (link)]. Inhibition% = [(OD control − OD treated)]/OD control] × 100%.
Seed cells on 96-well plates by 5000 cells/well and treat with oridonin, with, as indicated, final concentrations for a different duration. Fix cells in situ with 40% (w/v) cold Trichloroacetic acid (TCA). Let them sit for 5 min. Incubate plates at 4 °C for 1 h. Discard supernatant and gently wash plates 5 times with water, followed by an air-dry. Stain fixed cells with 0.4% (w/v) SRB with 1% acetic acid as solubilizer. After staining, wash with 1% acetic acid to remove the free dye and air-dry. Solubilize the bound stain with 100 mM Tris Base for reading. Read absorbance on a microplate reader at 515 nm [45 (link),46 (link)]. Inhibition% = [(OD control − OD treated)]/OD control] × 100%.