ViaKrome Fixable Viability Dyes: A new approach to dead cell discrimination for intracellular multicolor flow cytometry application

Abstract The ability to stain live cells with a viability dye and preserve that staining pattern after fixation is critical for intracellular immunophenotyping by flow cytometry. Excluding dead cells from the data allows cleaner separation and identification of cell populations. All commercially available viability dyes are based on NHS ester activated functional groups crosslinking with primary amines on proteins. These functional groups are susceptible to hydrolysis, necessitating the use of non-aqueous solvents, and additional reagent handling steps to protection from water vapor, such as aliquoting the stock solution and reducing freeze-thaw cycles. Here, we propose a new thiol reactive fluorescent dye that covalently binds free thiols present on cellular proteins at a neutral pH. Live cells, impermeable to the FVD, will be weakly stained by the covalent binding of proteins express on the cell surface while dead cells whose membranes have lost structural integrity, will be strongly stained by the covalent binding of intracellular and membrane expressed proteins. Contrary to current dyes, the ViaKrome dyes are soluble in aqueous solution and do not require DMSO for re-suspension. Hence, we show that the stability of dye ensure robust and reproducible experimental data. To further increase the ease of use, this new thiol based chemistry is available in a range of dyes excited by the common lasers used in flow cytometry. This choice provides flexibility for multicolor panel design. The compatibility of the ViaKrome dyes, i.e ViaKrome 405, ViaKrome 561, ViaKrome 638 and ViaKrome 808 will be presented in different workflow set-up such as 18-colors immunphenotyping panels and functional assays using PBMCs and whole blood.