Rosa26-INDIA Identifies Early Apoptotic B Cells Before Macrophage-Mediated Efferocytosis

Apoptotic cells are removed by efferocytosis, raising the possibility that in vivo apoptotic cell frequencies underestimate cell death. Rosa26-INDIA is an apoptosis reporter that detects activated caspase-3 (Casp3) via Förster resonance energy transfer (FRET) loss, but the relationship between Casp3 activation, phosphatidylserine exposure, membrane permeabilization, and efferocytic clearance remains unclear. We combined live-cell imaging and macrophage depletion to evaluate apoptotic B cell detection by Rosa26-INDIA. Live-cell imaging revealed that FRET loss preceded loss of detectable INDIA, Annexin-V positivity, and DAPI uptake. Loss of detectable INDIA occurred on average 6 and 12 min before Annexin-V and DAPI positivity, respectively, indicating that Rosa26-INDIA preferentially identifies early apoptotic cells. Consistently, macrophage depletion had limited effects on Rosa26-INDIA-based detection of apoptotic B cells under both steady-state and dexamethasone-induced conditions. These findings demonstrate that Rosa26-INDIA reports early apoptosis and suggest that macrophage-mediated efferocytosis does not substantially limit Rosa26-INDIA-based detection of apoptotic B cells under the conditions examined.