Apoptosis of LO2 and HepG2 cells induced by PIX was detected by using Annexin V-FITC detection kits (BD Biosciences). Briefly, cells treated with PIX (16 to 32 μg/mL) for 24 h under standard conditions (DMEM with 10% FBS, 5% CO2, 37°C) were collected by centrifugation and resuspended gently in 100 μL of 1× Annexin V binding buffer at a concentration of 1 × 106 cells/mL. Annexin V-FITC (5 μL) and PI (5 μL) were added to the suspension. After incubating at room temperature for 15 min in the dark, the stained cells were analyzed by flow cytometry (71 (link)).
For CLSM observation, RAW264.7 cells were cultured in standard conditions (DMEM supplemented with 10% FBS, 5% CO2, 37°C) supplemented with PIX (32 to 128 μg/mL) in humidified incubators. After incubating for 24 h, the cells were collected and stained with Annexin V-FITC detection kits. Then, 20 μL of the suspension was moved onto a glass slide, allowing it to be air dried and imaged.
For CLSM observation, RAW264.7 cells were cultured in standard conditions (DMEM supplemented with 10% FBS, 5% CO2, 37°C) supplemented with PIX (32 to 128 μg/mL) in humidified incubators. After incubating for 24 h, the cells were collected and stained with Annexin V-FITC detection kits. Then, 20 μL of the suspension was moved onto a glass slide, allowing it to be air dried and imaged.
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