Cellular senescence is a major driver of aging and age-related diseases. Quantification of senescent cells remains challenging due to the lack of senescence-specific markers and generalist, unbiased methodology. Here, we describe the Fully-Automated Senescence Test (FAST), an image-based method for the high-throughput, single-cell assessment of senescence in cultured cells. FAST quantifies three of the most widely adopted senescence-associated markers for each cell imaged: senescence-associated β-galactosidase activity (SA-β-Gal) using X-Gal, proliferation arrest via lack of 5-ethynyl-2’-deoxyuridine (EdU) incorporation, and enlarged morphology via increased nuclear area. The presented workflow entails microplate image acquisition, image processing, data analysis, and graphing. Standardization was achieved by i) quantifying colorimetric SA-β-Gal via optical density; ii) implementing staining background controls; iii) automating image acquisition, image processing, and data analysis. We show that FAST accurately quantifies senescence burden and is agnostic to cell type and microscope setup. Moreover, it effectively mitigates false-positive senescence marker staining, a common issue arising from culturing conditions.

Using FAST, we compared X-Gal with fluorescent C12FDG live-cell SA-β-Gal staining on the single-cell level. We observed only a modest correlation between the two, indicating that those stains are not trivially interchangeable. Finally, we provide proof of concept that our method is suitable for screening compounds that exacerbate or mitigate senescence burden (i.e. senescence inducers and senolytics, respectively). This method will be broadly useful to the aging field by enabling rapid, unbiased, and user-friendly quantification of senescence burden in culture, as well as facilitating large-scale experiments that were previously impractical.

A Fully-Automated Senescence Test (FAST) for the high-throughput quantification of senescence-associated markers

The establishment of a local lunar time standard or a shared and openly
accessible reference timing signal can greatly enhance the positioning, navigation, and timing (PNT) capabilities of lunar missions without the need for direct links to Earth or precision instruments to be a requirement for every mission to the Moon. This paper proposes the concept of a local lunar time standard that can be accessed using technology that is likely to be included in most lunar missions for nominal activities. Even if the local lunar time standard exhibits drift or variations from terrestrial time, its existence would be  a  signiﬁcant  step  towards  lunar coordination.  Unlike  terrestrial synchronized time, a fully usable lunar reference time can be implemented proactively before the activity that necessitates it occurs. This paper discusses the potential beneﬁts and challenges of establishing a local lunar time standard and its implications for lunar missions, including its compatibility with existing lunar initiatives such as LunaNet, Moonlight, and upcoming lunar missions. The proposal highlights the opportunities and considerations for stakeholders in the cislunar ecosystem, including options for missions to ignore, participate in seeding, or consume the proposed timekeeping protocol. The proposal is expected to contribute to the evolving landscape of timekeeping technologies and methods in cislunar space, and stimulate further research and discussion on this topic.