Figure 1. Schematic illustration of the annexin V binding assay.

Preparation of a negative control without DAPI and annexin V staining, a control with DAPI-stained cells, and cells stained with DAPI and annexin V conjugated to Alexa Fluor 568 for 10 min on ice with TBSS with or without calcium, respectively. The negative control without staining is used to assess the level of background fluorescence in the sample. This is important because even in the absence of a fluorescent stain, there may still be some level of background fluorescence present due to autofluorescence or other sources. DAPI staining (at a final concentration of 10 μg/mL) is used for visualization of nuclei and cell counting. Non-specific binding of Ca²⁺-dependent annexin V is tested in the same assay by using TBSS without Ca²⁺.