A β-galactosidase probe for the detection of cellular senescence by mass cytometry

Abstract

Mass cytometry (MC) is a powerful method that combines the cellular resolution of flow cytometry with the isotopic resolution of inductively coupled plasma mass spectrometry (ICP-MS). This combination theoretically allows for the simultaneous quantification of >80 different parameters at the single cell level, in turn allowing for the deep profiling of heterogeneous cell populations. The majority of available reagents for MC are antibodies labeled with heavy metal isotopes, allowing for the quantification of static biomarkers. To complement these reagents, we aim to develop small molecule reporters of cellular metabolism that are compatible with MC. Here we report a probe of β-galactosidase activity capable of detecting cellular senescence. The galactoside probe contains a tellurophene reporter group and, when hydrolyzed, generates a quinone alkide. This reactive alkylating agent forms covalent tellurophene bearing conjugates with local nucleophiles, allowing for the quantification of β-galactosidase activity in individual cells. Difluoromethyl and monofluoroethyl quinone alkide generating warheads were examined for their activities and compared in vitro and in vivo. We showed that the difluoromethyl derivative gave higher tellurium labelling in vitro and that the quinone methide was more reactive towards thiols than amines. In vivo the difluoromethyl derivative successfully labeled senescent cells with comparable selectivity to the commonly used fluorescent senescence probe CFDG.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…As such, the EpiTOF (Epigenetic landscape‐profiling using cytometry by time‐of‐flight) Ab panel allows the assessment of different classes of histone modifications and variants. Functional probes available for mass cytometry include 5‐iodo‐2‐deoxyuridine for assessing cell proliferation , enzymatic activity , and a tellurium‐based hypoxia probe . Wheat germ agglutinin (WGA) and osmium tetroxide staining were proposed as a proxy for cell size in mass cytometry , besides ASCQ‐Ru for cell volume respectively, which, in conjunction with the Celltracer software has been used to correct for confounding cell size effects in signaling studies .…”

Hallmarks of senescence and aging

“…Recently a new method for the determination of lipofuscin in liquid samples of stressed or damaged cells was introduced ( 85 ). Mass cytometry method, as a method that combines flow cytometry and mass spectrometry, enables the simultaneous quantification of numerous cellular parameters (SA-β-GAL) at single-cell resolution ( 86 ). Also, among potential predictors of biological age could be included the degree of methylation of DNA, transcriptomic predictors, proteomic predictors, metabolomics-based predictors, and composite biomarker predictors ( 87 ).…”

An Iodinated DAPI‐Based Reagent for Mass Cytometry

“…To incorporate cellular phenotype reporters and cell‐type barcoding in parallel with antibody panels, small molecules bearing heavy elements were pursued for MC and IMC™ applications. This led to the development of numerous scaffolds, such as iridium and rhodium organometallic intercalators for cell enumeration, [29] bifunctional palladium and indium chelates for cell barcoding, [30,31] cisplatin for live/dead cell staining, [32] osmium and ruthenium tetroxide salts for cell barcoding and cell size discrimination, [33] bulky alkyl‐derivatives of chelators for staining bacteria [34] and eukaryotic organelles, [58] 5‐iodo‐2‐deoxyuridine (IdU) for cell cycle analysis, [35] and a collection of organotelluriums for a variety of applications including live cell barcoding in heterogenous suspensions [36] and organoids, [37] amplification of low abundance antigens in IMC™; [38] and monitoring biological states such as hypoxia, [39,40] senescence associated β‐galactosidase activity, [41] and protein synthesis turnover [42] . With the updated collection of MC and IMC™ reagents, 40–50 parameters can now be measured simultaneously from individual cells.…”