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.

Cells were adjusted to a concentration of 10×10³ cells/µL using PBS. A 100 µL aliquot of the cell suspension was placed in a test tube. Cells were stained with 10 µL of PE-conjugated anti-CD34 (BD, Biosciences) and 5 µL of PC7-conjugated anti-CD45 (BD, Biosciences) for 15 min at room temperature in the dark. Erythrocytes were lysed using an ammonium chloride (NH₄Cl)-based buffered solution. After washing, the cells were adjusted to a concentration of 1×10⁶ cells in 100 µL of the annexin-V binding buffer (BD, Biosciences) and stained with 10 µL of FITC-conjugated annexin-V (BD, Biosciences) for 15 min on ice in the dark. After adding 10 µL of peridinin-chlorophyll-protein complex (PerCP)-binding 7-AAD and 380 µL of annexin-V binding buffer, cells were analyzed by flow cytometry (FC-500; Beckman Coulter). From each sample, 50,000 events were acquired and analyzed from among the live cell population (negative for 7-AAD).