Table 2.

Multi-fluorescence properties of double-stained cells collected in a same area with the explanation of the underlying cellular mechanism.

	Dyes	Fluorescence	Multi-fluorescence		Status of cellular mechanism
I. Membrane integrity	Syto9 (or SYBR-I, Syto13)/PI	Green/Red	Syto9+	PI−	Intact cells Viable cells
			Syto9−	PI+	Permeabilized cells Dead cells
			Syto9+	PI+	Partially-permeabilized cells Injured cell populations
			Syto9−	PI−	Unstained debris
	References: Possemiers et al., 2005; Pianetti et al., 2008; Kim et al., 2009; Muñoz et al., 2009; Spilimbergo et al., 2010; Martínez-Abad et al., 2012; Choi et al., 2013; Tamburini et al., 2013; Booyens and Thantsha, 2014; Fernandes et al., 2014; Manoil et al., 2014; Pal and Srivastava, 2014; Tamburini et al., 2014; Boda et al., 2015; Ferrentino et al., 2015; Freire et al., 2015; Li H. et al., 2015; Li W. et al., 2015; Muriel-Galet et al., 2015
	TO/PI	Green/Red	TO+	PI−	Intact cell membranes
			TO−	PI+	Permeabilized cell membrane
			TO+	PI+	Cell membrane damaged, slightly permeabilized
			TO−	PI−	Damage of DNA and RNA while the cell may still be intact
	References: Surowsky et al., 2014
II. Membrane integrity/physiological state
A. Enzyme activity	PI/cFDA	Red/Green	PI+	cF−	Esterase activity not detectable, membrane compromised dead cells
			PI+	cF+	Viable injured or stressed cells with intracellular activity and minimally compromised membrane
			PI−	cF+	Active esterase, intact membrane viable cells
			PI−	cF−	Inactive esterase or cF extruded out of the cells, intact membrane, or unstained debris
	References: Ananta et al., 2004; Hayouni et al., 2008; Ananta and Knorr, 2009; Schenk et al., 2011; Ayari et al., 2013; Thabet et al., 2013; Surowsky et al., 2014; Hong et al., 2015; Combarros et al., 2016; Meng et al., 2016
B. Pump activity	Syto9/EB	Green/Red	(Syto9+ EB+ green and red high intensity)		Cells with efflux pump completely damaged
	References: Kim et al., 2009
C. Membrane potential	IP/DiBAC4	Red/Green	PI−	DiBAC4−	Unstained population of intact cells
			PI−	DiBAC4+	Depolarized cells
			PI+ PI+	DiBAC4− DiBAC4+	Population of permeabilized cells with different degrees of damage
	References: Novo et al., 2000; Wu et al., 2010a,b; Coronel-León et al., 2016; Grau-Campistany et al., 2016
	Syto40/DiBAC4	Blue/Green	Syto40+	DiBAC4+	Depolarized cells
			(blue and green high intensity)
	References: Duarte et al., 2015
	TO-PRO®3/DiOC2(3)	Far red/Green shift to red	TO-PRO®3+	DiOC2(3)+	Permeabilized and depolarized
			TO-PRO®3+	DiOC2(3)−	Permeabilized and polarized
			TO-PRO®3−	DiOC2(3)+	Non-permeabilized and depolarized
			TO-PRO®3−	DiOC2(3)−	Non-permeabilized and polarized
	References: Novo et al., 2000; Hammer and Heel, 2012
D. Respiratory activity	Syto40/CTC	Blue/red	Syto40+	CTC+	Cells with respiratory activity
			(blue and red high intensity)
	References: Duarte et al., 2015
	Syto9/CTC	Green/red	Syto9+	CTC+	Cells with respiratory activity
			(green and red high intensity)
	References: Ferreira et al., 2014
III. DNA/Antibody β-lactamase activity	Syto62/anti β -lactamase mAb AF488-DAM IgG	Red/Green	Syto62+	anti β-lactamase mAb AF488-DAM IgG+	Cells with β-lactamase activity ampicillin resistant
	References: Huang et al., 2016

PI, Propidium Iodide; TO, Thiazole Orange; cFDA, carboxyFluorescein DiAcetate; DiBAC₄, Bis-(1,3-DibutylBarbituric ACid) Trimethine Oxonol = bis-oxonol = BOX; DiCO₂, 3,3-DiethyloxaCarbocyanine Oxide; CTC, 5-Cyano-2,3-ditolyl Tetrazolium Chloride; EB, Ethidium Bromide.