Table 1.

Current and proposed criteria to define ApoBDs.

	Proposed
Current Criteria	Criteria	Rationale / discussion
Detection: Visualization of ApoBDs by light microscopy or electron microscopy [33–38]. Quantification of ApoBDs by flow cytometry [39–42].	For in vitro studies, visualization of ApoBD formation (i.e. morphological steps of apoptotic cell disassembly) by live time-lapse microscopy. Visualization of ApoBDs by electron microscopy only when appropriate. Monitor the level of apoptosis. For both microscopy and flow cytometry approaches to detect ApoBDs, criteria below (size, markers and other properties) should be considered.	Provides evidence that ApoBDs are indeed generated from cells undergoing apoptosis. Electron microscopy analysis is suitable to monitor ApoBDs in tissue samples [33,48,55], however, additional sample processing steps may alter ApoBD properties. Provides evidence that apoptosis did occur as this is the prerequisite for ApoBD formation. Provides evidence that the vesicles observed are indeed ApoBDs and not other types of extracellular vesicles.
Size: 1–5 μm in diameter [3,19–22,33–38].	The lower and upper limit of ApoBD diameter should be cell type dependent.	The size of ApoBDs will vary between cell types, likely due to differences in cell size and the mechanism of ApoBD formation.
Markers: PtdSer exposure [39,53], contains nuclear proteins and DNA [1–3,24,38,81,82], enrichment in calreticulin [28,62].	ApoBDs can be PtdSerIntermediate or PtdSerLow. Cell type-specific markers are retained on ApoBDs [57]. ApoBDs can be with or without DNA/nuclear proteins [45,53]. For immunoblot analysis of purified ApoBDs, a number of caspase 3/7-cleaved plasma membrane and cytoplasmic proteins should be monitored.	PtdSer exposure on ApoBDs is a spectrum. Cell type-specific markers are retained on ApoBDs but at a markedly reduced level compared to apoptotic and viable cells [57]. Whether DNA and nuclear proteins are distributed into ApoBDs is cell type dependent (likely due to differences in the mechanism of ApoBD formation) and not all ApoBDs contain these nuclear materials [45,53]. Although caspases could be active under non-apoptotic conditions, monitoring proteins that are cleaved by caspase 3/7 during apoptosis in purified ApoBDs provides additional evidence that the purified sample contain ApoBDs. It should be noted that determining the level of calreticulin will further validate the presence of ApoBDs [28,62].
Other properties: Lysed [67].	ApoBDs can be both intact and lysed (i.e. membrane permeabilized, leading to leakage of luminal material).	ApoBDs generated under in vitro conditions will become permeabilized over time.