Cell Separation by Countercurrent Centrifugal Elutriation: Recent Developments

Grosse, Jirka; Bauer, Thomas Johann; Eilles, Christoph; Grimm, Daniela

doi:10.1080/10826068.2011.602799

Cited by 12 publications

(9 citation statements)

References 108 publications

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“…PBMC were isolated by Ficoll-Paque density gradient centrifugation (GE Healthcare, Chalfont St. Giles, UK) from healthy buffy coats obtained from the Australian Red Cross. PBMC were further separated into three populations by counter-current elutriation using Beckman J-6M/E centrifuge equipped with a JE 5.0 rotor (Beckman Coulter, Pasedena, CA, USA; [ 70 ]). The three fractions were isolated at rates of 12 (small lymphocytes), 16 (large lymphocytes) and 20 (DC/Monocytes fractions) ml/min.…”

Section: Methodsmentioning

confidence: 99%

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

et al. 2015

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BackgroundCombination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4+ T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4+ T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4+ T-cells co-cultured with CD11c+ myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4+ T-cells, and examined potential cell interactions that may be involved using RNA-seq.ResultsmDC (CD1c+), SLAN+ DC and CD14+ monocytes were most efficient in stimulating proliferation of CD4+ T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4+ T-cells following HIV-1 infection of APC-T cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4+ T-cells. Gene expression analysis, comparing the CD1c+ mDC, SLAN+ DC and CD14+ monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4+ T-cells via cell–cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene).ConclusionsAPC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14+ monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4+ T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV-1 latency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0204-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

et al. 2015

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“…Counter-flow centrifugal elutriation (CCE) is a reliable, effective, and widely-used method to synchronize cells for cell cycle research (Bauer, 1999;Davis et al, 2001;Banfalvi, 2008Banfalvi, , 2011Coulais et al, 2012;Grosse et al, 2012;Kume, 2016;Delgado et al, 2017), but how to optimize the procedure by determining specific parameters for particular applications remains challenging. There are three key parameters that collectively determine the results of elutriation: centrifugal force, collection flow rate, and collection volume.…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Nan

Niu

et al. 2021

Front. Cell Dev. Biol.

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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.

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“…Readers can refer to reviews by Grosse and colleagues (79) for information about macroscale CCE applications. Banfalvi (80) has used CCE for cell synchronization because CCE does not disturb cell-cycle progression, and highly synchronized cell populations can be obtained especially in the G 1 phase, and early and mid-S phase.…”

Section: Centrifugal Methods (Operation At High Reynolds Numbers): Hymentioning

confidence: 99%

Large-Volume Microfluidic Cell Sorting for Biomedical Applications

Warkiani

Wu²,

Tay

et al. 2015

Annu. Rev. Biomed. Eng.

102

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Microfluidic cell-separation technologies have been studied for almost two decades, but the limited throughput has restricted their impact and range of application. Recent advances in microfluidics enable high-throughput cell sorting and separation, and this has led to various novel diagnostic and therapeutic applications that previously had been impossible to implement using microfluidics technologies. In this review, we focus on recent progress made in engineering large-volume microfluidic cell-sorting methods and the new applications enabled by them.

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Cell Separation by Countercurrent Centrifugal Elutriation: Recent Developments

Cited by 12 publications

References 108 publications

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4+ T-cells

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

Large-Volume Microfluidic Cell Sorting for Biomedical Applications

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