Abstract
Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging. multFYLM captures approximately one thousand rod-shaped fission yeast cells from up to six different genetic backgrounds or treatment regimens. The immobilized cells are fluorescently imaged for over a week, while the progeny cells are removed from the device. The resulting datasets yield high-resolution multi-channel images that record each cell's replicative lifespan. We anticipate that the multFYLM will be broadly applicable for single-cell whole-lifespan studies in the fission yeast (Schizosaccharomyces pombe) and other symmetrically-dividing unicellular organisms.
Highlights
[Background] Cellular aging results in the cumulative decline of cellular function that eventually leads to mortality
The replicative lifespan (RLS) of a cell is defined as the number of daughters produced by a mother cell over the course of its life (Henderson and Gottschling, 2008; Sutphin et al, 2014)
RLS studies have greatly expanded our understanding of cellular aging in mitotically active cells
Summary
Shaking incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4333) 2. Mini vortexer (Fisher Scientific, catalog number: 02-215-365) Note: This product has been discontinued. The first step is to generate a patterned mold, which can be used to cast devices in elastomeric silicone (PDMS) Such molds, or ‘master’ structures are created on silicon wafers, using UV lithography to deposit patterns on the surface in an epoxy resin (SU-8). The patterns are dictated by masks, which restrict the ability of a UV light source to cross-link the resin Their alignment is critical to the proper patterning on the wafer, as features of the final master are contained on each of the two masks. Place the wafer directly on a hotplate with the polished surface face up for 20 min at 200 °C. The parameters here should only be used as a guideline
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