Annexin 5 binding buffer 1x

2.5X10⁵ BMMC/0.25 mL of complete medium were stimulated with L.m at different MOI (1:1, 10:1, 100:1) or stimulated with LLO (125, 250, 500, 1000 ng/mL) for 24 h. Then cells were washed with 1 mL of Annexin V binding Buffer 1X (Invitrogen, USA) and stained with 1 μg/mL Annexin V (BioLegend, USA) and 0.5 μg/mL propidium iodide (eBioscience, USA). After staining, cell viability was measured by flow cytometry (Supplementary Figure 2).

In total, 1 × 10⁶ DPSCs were labeled with 180 μg/mL of iron oxide and transfected with 1 µL/mL of lipofectamine to determine apoptosis by flow cytometry. In brief, they were trypsinized and transferred into a falcon tube. Using a cold PBS solution, the cells were centrifuged at 200× g for 5 min. The supernatant was removed, and the cell pellet was washed two times with Annexin V-PE early apoptosis detection kit I (BD Biosciences, Billerica, MA, USA) suspended in 0.1 mL of Annexin V binding buffer 1X (Invitrogen, USA). Then, addition of 5 µL of Annexin V-PE (Invitrogen, Carlsbad, CA, USA) and 5 μL of 7-Aminoactinomycin D 7-AAD (Invitrogen, USA) took place. They were slowly vortexed and then left for 15 min at room temperature. In total, 400 μL of Annexin V binding buffer (1×) was added as attachment buffer and was further read by a Fluorescence-Activated Cell Sorting (FACS) Calibur flow cytometer (BD Biosciences, Germany) for an hour, and was finally analyzed by the Flow Jow software. The amounts of early apoptosis were determined as the population percentage of Annexin V+/7-AAD, and late apoptosis as the population percentage of Annexin V+/7-AAD + labeled cells with 180 μg/mL of iron oxide and transfected with 1 µ/mL of lipofectamine.