Annexin binding buffer

The mProx24 cells were treated with CsA or CsA plus 5‐ALA/SFC and then were plated in six‐well plates. After incubation for 12 and 24 h, the cells were stained with FITC‐Annexin V (Biolegend, Tokyo, Japan) and propidium iodide (PI) and washed with PBS. They were then centrifuged at 270 g. for 5 min. The cells were resuspended at a density of 1 × 10⁶ cells per mL with Annexin‐binding buffer (Biolegend). To every 100 μL of cell suspension, 5 μL of FITC‐Annexin V and 2 μL of PI were added. The cells were incubated at room temperature for 20 min. Then, 500 μL of 1× Annexin‐binding buffer was added, and the cells were analyzed immediately by flow cytometer (Gallios; Beckman Coulter, Tokyo, Japan).

Cells were seeded at 1×10⁵ cells/10 ml in 6-well plates and incubated overnight. Live cells were collected and washed twice by ice-cold PBS. cells were stained with Annexin V fluorescein dye and propidium iodide (PI) at room temperature in dark for 15 min. cells were subsequently resuspended in 400 μl Annexin-binding buffer (Beckman Coulter, Inc., Brea, CA). The stained cells were kept on ice and analyzed by Beckman Flow Cytometers.

Mouse blood cells were washed twice using Ringer’s solution supplemented with 5 mM calcium chloride. To detect FITC annexin V-positive cells, the erythrocytes were suspended in an annexin-binding buffer (BD Pharmingen, San Diego, United States) with FITC annexin V (1:200 dilution, BD Pharmingen, San Diego, United States) and incubated for 15 min at room temperature. Well mixing was performed by pipetting. Finally, erythrocytes were diluted five times in the annexin-binding buffer before analysis in the FACSVerse flow cytometer (Beckman Coulter, CytoFlex S, United States) at an excitation wavelength of 488 nm (blue laser) and emission wavelength of 530 nm.