Flow cytometry-based viability staining: an at-line tool for bioprocess monitoring of Sulfolobus acidocaldarius

AMB Express

Table 1 Overview of the investigated fluorescent dyes

Dye	Ex. λ_max¹ [nm]	Em. λ_max² [nm]	Fluorescence color	Permeability*	Mode of interaction	For detection of
Acridine orange (AO)	500	526	green	permeable	DNA/RNA	viable and non-viable cells
SYTO™ 9	485	500	green	permeable	DNA/RNA	viable and non-viable cells
RH414	532	716	red	permeable	cell membrane	viable and non-viable cells
Concanavalin A-rhodamine	545	570	red	impermeable	cell membrane	viable and non-viable cells
Fluorescein diacetate (FDA)	485	520	green	permeable	enzymatic fluorophore generation	viable cells
DiBAC₄(3)	493	516	green	impermeable	positively charged or hydrophobic regions	non-viable cells
Propidium iodide (PI)	535	617	red	impermeable	DNA/RNA	non-viable cells
7-AAD	546	647	red	impermeable	DNA, G-C rich regions, RNA	non-viable cells

Acridine orange (AO) was obtained from Carl Roth (Germany). SYTO™ 9 and 7-Aminoactinomycin D (7-AAD) were purchased from Thermo Fisher Scientific (USA). RH414 [N-(3-Triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl) pyridinium dibromide] as well as DiBAC₄(3) [Bis- (1,3-dibutylbarbituric acid) trimethine oxonol] were obtained from AnaSpec (USA). Concanvalin A—rhodamine was supplied by Vector Laboratories (USA) while fluorescein diacetate (FDA) and propidium iodide (PI) was purchased from Sigma Aldrich (USA)
¹Ex λ_max: maximum excitation wavelength
²Em λ_max: maximum emission wavelength
*here permeability describes the capability of a dye to enter intact (uncompromised) cells