An Optimized and Versatile Counter-Flow Centrifugal Elutriation Workflow to Obtain Synchronized Eukaryotic Cells.

Yongqiang Liu,Geoffrey M Kapler,Junhua Niu,Shan Gao,Bei Nan

doi:10.3389/fcell.2021.664418

Abstract

Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, Tetrahymena thermophila as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase Tetrahymena as measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.

Highlights

Synchronization of cell populations is a powerful tool for studying cell cycle regulated events, such as organelle biogenesis, DNA replication, chromosome segregation and the establishment of epigenetic marks on daughter chromosomes (Kolb-Bachofen and Vogell, 1975; Banfalvi, 2011; Jiang et al, 2014, 2019; Sandoval et al, 2015; Delgado et al, 2017; Li et al, 2020)
The two most commonly used separation methods are counter-flow centrifugal elutriation (CCE) and fluorescence-activated cell sorting (FACS) (Bauer, 1999; Banfalvi, 2008; Delgado et al, 2017), due to their minimal effect on cell cycle progression
The cell cycle progression and synchrony of each elutriated fraction are assessed by flow cytometry, the results of which are affected by the adhesion between nuclei

Summary

Introduction

Synchronization of cell populations is a powerful tool for studying cell cycle regulated events, such as organelle biogenesis, DNA replication, chromosome segregation and the establishment of epigenetic marks on daughter chromosomes (Kolb-Bachofen and Vogell, 1975; Banfalvi, 2011; Jiang et al, 2014, 2019; Sandoval et al, 2015; Delgado et al, 2017; Li et al, 2020). Most widely used approaches are based on one of two distinct strategies for obtaining a homogeneous cell population: transient cell cycle arrest or physical separation. Treated cells are arrested at a particular stage of the cell cycle and allowed to progress to the stage synchronously upon release of the block. These manipulations, may perturb cell physiology and can alter the behavior of the cell populations in an unpredictable manner (Cooper, 2003; Banfalvi, 2008). The two most commonly used separation methods are counter-flow centrifugal elutriation (CCE) and fluorescence-activated cell sorting (FACS) (Bauer, 1999; Banfalvi, 2008; Delgado et al, 2017), due to their minimal effect on cell cycle progression

Methods

Results

Conclusion