Check chelator carryover
Check dissociation, wash and storage buffers for EDTA or other calcium chelators. A calcium-containing final buffer does not eliminate interference from carryover. Transfer samples as specified in the product instructions; do not substitute ordinary calcium-free PBS for binding buffer.
Distinguish buffer effects from cell state
For unexpected signals, assess the binding buffer with consistently handled controls and review membrane-integrity readouts. Resolve differences in collection or waiting times before attributing changes to Annexin V binding.
Avoid compensating for unknown carryover by adding calcium
Effective EDTA and calcium concentrations depend on carryover volume and buffer composition. Remove residual chelator and transfer into binding buffer using a validated workflow rather than repeatedly adding calcium to compensate for unknown carryover.