A platinum-based covalent viability reagent for single-cell mass cytometry

Abstract

In fluorescence-based flow cytometry, cellular viability is determined with membrane-impermeable fluorescent reagents that specifically enter and label plasma membrane-compromised nonviable cells. A recent technological advance in flow cytometry uses antibodies conjugated to elemental metal isotopes, rather than to fluorophores, to allow signal detection by atomic mass spectrometry. Unhampered by the limitations of overlapping emission fluorescence, mass cytometry increases the number of parameters that can be measured in single cells. However, mass cytometry is unable to take advantage of current fluorescent viability dyes. An alternative methodology was therefore developed here in which the platinum-containing chemotherapy drug cisplatin was used to resolve live and dead cells by mass cytometry. In a 1-min incubation step, cisplatin preferentially labeled nonviable cells from both adherent and suspension cultures, resulting in a platinum signal quantifiable by mass cytometry. This protocol was compatible with established sample processing steps for intracellular cytometry. Furthermore, the live/dead ratios were comparable between mass- and fluorescence-based cytometry. Importantly, although cisplatin is a known DNA-damaging agent, a 1-min "pulse" of cisplatin did not induce observable DNA damage or apoptotic responses even within 6-h post-exposure. Cisplatin can therefore be used as a viability reagent for a wide range of mass cytometry protocols.

Figure 1

Cisplatin as a viability reagent for mass cytometry. (A) In a one-minute “pulse” staining protocol, cisplatin preferentially reacts with protein in dead cells. (B) As a basis for comparison, an aliquot of partially heat-killed KG-1 cells was labeled with an amine-reactive fluorescent viability stain (Aqua). (C) Additional aliquots of partially heat-killed KG-1 cells were labeled with cisplatin at the concentrations shown and analyzed by mass cytometry.

Figure 2

Cisplatin as a viability indicator for suspension and adherent cell lines. Cisplatin viability labeling was performed at 10, 50 and 250 µM final concentrations for suspension (Jurkat, KG-1) and adherent (HeLa, OVCAR3) cell lines. For all cell lines the percentage of dead cells was comparable as determined by mass cytometry and Trypan blue exclusion.

Figure 3

Reproducibility and dynamic range of the cisplatin viability reagent by mass cytometry. Triplicate samples of HL-60 cells spiked with varying percentages of dead cells ranging from 0–40% were analyzed by mass cytometry using the cisplatin viability reagent. The average percent of dead cells in each sample with their standard deviation are shown. Mass cytometry measurements of cisplatin labeling reproducibly determined the percentage of dead cells over the whole analysis range.

Figure 4

Cisplatin-high PBMCs were unable to signal in response to pervanadate stimulation. (A) Cryopreserved peripheral blood mononuclear cells were labeled with cisplatin, 25 µM, final concentration for one minute and treated with PVO₄, 125 µM, final concentration for 15 minutes. An antibody that cross-reacted with phosphorylated SLP-76 (Tyr128) and SLP-65 (Tyr72) was used as an indicator of intracellular signaling. (B) Histogram overlays shown for pSLP-76/65 in untreated (grey) and vanadate-treated (white) populations. (R1) includes all cells, (R2) only cisplatin low and (R3) only the cisplatin high population.

Figure 5

Cisplatin viability labeling did not induce DDR or apoptosis. When compared with untreated cells, no induction of phosphorylated Ser139 on H2AX (pH2AX) or cleaved PARP (cPARP) was apparent in KG-1 cells incubated for 30, 60, 120, 240 and 360 minutes in cisplatin-free media following a 1-minute “pulse” treatment with cisplatin, 25 µM final concentration. A separate aliquot of cells was treated continuously with 20 µM etoposide for 9 hrs as a positive control for the pH2AX and cPARP antibodies.