Annexin 5 binding buffer

Cells were seeded at 1×10⁶ cells in 2.5 ml RPMI 1640 medium containing 10% FBS per well in 6-well plates for 24 h at 37°C prior to exposure to the corresponding treatment. Following incubation, the cells in each group were exposed to the corresponding treatment for an additional 24 h, and the supernatant medium from each well was transferred in a separate pre-labeled centrifuge tube to collect non-adherent cells. Adherent cells from the same well were then trypsinized and transferred to the same centrifuge tube containing the non-adherent cells. Cells in each centrifuge tube were then centrifuged at 2,000 × g at 4°C for 5 min, and the supernatant was discarded. The cells were re-suspended in 100 µl Annexin V binding buffer (Dojindo Molecular Technologies, Inc.). In total, 5 µl FITC-Annexin V and 5 µl PI (both from Dojindo Molecular Technologies, Inc.) were added and the cells were incubated at room temperature for 15 min in the dark. Subsequently, 400 µl Annexin V binding buffer was added and flow cytometry was performed on a Beckman flow cytometer (Beckman Coulter, Inc., Brea, CA, USA). Cells were considered to be apoptotic if they were Annexin V⁺/PI⁻ (early apoptotic) and Annexin V⁺/PI⁻ (late apoptotic). At least 10,000 events were recorded and the data were evaluated using Kaluza 1.3 software (Beckman Coulter, Inc.) for each analysis.

To determine the level of early-stage apoptosis, CMECs were rinsed twice with ice-cold PBS. After trypsinization, CMECs were washed twice with PBS and centrifuged at 1000 rpm for 5 min. Subsequently, cells were resuspended in 100 μL Annexin V binding buffer (Dojindo Laboratories, Shanghai, China) containing 5 μL Annexin V-FITC plus 5 μL propidium iodide (PI), before incubation at room temperature (RT) in the dark for 15 min. After resuspending in 400 μL Annexin V binding buffer, the samples were immediately measured by flow cytometry (Accuri™ C6, BD Biosciences, CA, USA).