Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

Tau assembly propagation from the extracellular to intracellular space of a cell may underlie neurodegenerative tauopathies. The first step involves tau binding to heparan sulfate proteoglycans on the cell surface, followed by macropinocytosis. Pathological tau assemblies are thought to exit the vesicular compartment as seeds for replication in the cytoplasm. Tau uptake is highly efficient, but only ~1-10% of cells that take up aggregates exhibit seeding. To investigate the basis for this observation, we used fluorescently tagged full-length (FL) tau fibrils added to native U2OS cells, and biosensor cells expressing FL tau or repeat domain fused to mClover (Clo). FL tau-Clo bound tubulin, but seeds triggered its aggregation in multiple locations simultaneously in the cytoplasm, generally independent of visible exogenous aggregates. Most exogenous tau trafficked to the lysosome, but imaging revealed a small percentage that slowly and steadily accumulated in the cytosol. Intracellular expression of Gal3-mRuby, which binds intravesicular galactosides and forms puncta upon vesicle rupture, revealed no evidence of vesicle damage following tau exposure. In fact, most seeded cells had no evidence of lysosome rupture. However, live cell imaging indicated that cells with pre-existing Gal3-positive puncta exhibited seeding at a slightly higher rate than the general population, indicating a potential role for vesicle instability as a predisposing factor. Clearance of tau seeds occurred rapidly in both vesicular and cytosolic fractions. Bafilomycin inhibited vesicular clearance, whereas MG132 inhibited cytosolic clearance. Tau seeds that enter the cell thus have at least two fates: lysosomal clearance that degrades most tau, and entry into the cytosol, where seeds replicate, and are cleared by the proteasome.

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“…Fractionation was guided by published protocols (8)(9). Cells were trypsinized and resuspended in 5mL of culture medium.…”

Section: Discussionmentioning

confidence: 99%

Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Kolay

Vega

Dodd

et al. 2022

Preprint

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show abstract

“…Fractionation was guided by published protocols (22,23). Cells were trypsinized and resuspended in 5 ml of culture medium.…”

Section: Cellular Fractionationmentioning

confidence: 99%

The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification

Kolay

Vega

Dodd

et al. 2022

Journal of Biological Chemistry

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“…Then the upper medium is removed carefully, followed by the injection of the fresh medium. However, the centrifugal cell medium exchange method is time‐consuming [44–48]; thus, it may change the state of cells and lead to a decrease in biological activity [44,49–52]. Moreover, this method cannot be used for micro‐volume cell samples due to its low harvest rate.…”

Section: Introductionmentioning

confidence: 99%

Automatic medium exchange for micro‐volume cell samples based on dielectrophoresis

Zhao

Shi

et al. 2021

Electrophoresis

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Cell medium exchange is a crucial step for life science and medicine. However, conventional cell medium exchange methods, including centrifuging and filtering, show limited ability for micro‐volume cell samples such as circulating tumor cell (CTC) and circulating fetal cell (CFC). In this paper, we proposed an automatic medium exchange method for micro‐volume cell samples based on dielectrophoresis (DEP) in microfluidic chip. Fresh medium and cell suspension were introduced into the microfluidic channel as the laminar flow. Plane stair‐shaped interdigital electrodes were employed to drive the cells from the cell suspension to fresh media directly by DEP force. Additionally, we characterized and optimized the cell medium exchange according to both the theory and experiments. In the end, we achieved a 96.9% harvest rate of medium exchange for 0.3 μL samples containing micro‐volume cells. For implementing an automatic continuous cell medium exchange, the proposed method can be integrated into the automatic cell processing system conveniently. Furthermore, the proposed method is a great candidate in micro‐volume cell analysis and processing, cell electroporation, single cell sequencing, and other scenarios.

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Cell Fractionation of U937 Cells in the Absence of High-speed Centrifugation

Cited by 5 publications

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Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

Dual fates of exogenous tau seeds: lysosomal clearance vs. cytoplasmic amplification

The dual fates of exogenous tau seeds: Lysosomal clearance versus cytoplasmic amplification

Automatic medium exchange for micro‐volume cell samples based on dielectrophoresis

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