Ethanol

BV2 microglia were collected after OGD/R. Total RNA was isolated by treating the cells with 500 mL of RNAiso or TRIzol (Takara, Tokyo, Japan) and 100 mL of trichloromethane (Sinopharm) for 5 minutes and then centrifuging the solution for 15 minutes. The supernatant was diluted in an equal volume of isopropanol, mixed for 10 minutes, and centrifuged for 10 minutes. Afterward, 1 mL of 75% ethanol (Sinopharm) was added to the mixture, which was then centrifuged for 5 minutes. The pellet was then resuspended in RNase-free H₂O (Takara), and the mixture was centrifuged at 12,000 × g, 4°C. Next, 1000 ng of purified total RNA was subjected to reverse transcription at 37°C for 15 minutes, then 5 seconds at 85°C with PrimeScript RT Master Mix (Takara). SYBR Premix Ex Taq (Takara) was used for quantitative polymerase chain reaction (qPCR) in a 20 μL reaction, using the reverse-transcribed cDNA as a template. The primer sequences are shown in Additional Table 1. A qPCR system (AB7500, Milwaukee, WI, USA) was used to perform the reaction at 95°C for 30 seconds followed by 45 cycles of 95°C for 15 seconds, 55°C for 5 seconds, and 72°C for 5 seconds. On the basis of the 2^–ΔΔCt method (Livak and Schmittgen, 2001), the relative expression of mRNA was calculated.

The cells in the seven study groups were plated in six-well plates and treated using the protocol described above. When the cell concentration was 1×10⁶ cells/ml, the medium was replaced with cold 75% ethanol (Sinopharm Chemical Reagent Co., Ltd, Beijing, China) to fix the cells at 4°C overnight. Then, 10 μL of Annexin V− fluorescein isothiocyanate (FITC) binding buffer (Sigma-Aldrich, St. Louis, MO, USA) and 5 μL of propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO, USA) were added to each well to incubate cells for 20 min avoiding light. The apoptosis rate of each well was detected using a flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).