Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry

Leelatian, Nalin; Doxie, Deon B.; Greenplate, Allison R.; Mobley, Bret; Lehman, Jonathan M.; Sinnaeve, Justine; Kauffman, Rondi M.; Werkhaven, Jay A.; Mistry, Akshitkumar M.; Weaver, Kyle D.; Thompson, Reid C.; Massion, Pierre P.; Hooks, Mary; Kelley, Mark C.; Chambless, Lola B.; Ihrie, Rebecca A.; Irish, Jonathan M.

doi:10.1002/cyto.b.21542

Cited by 60 publications

(70 citation statements)

References 28 publications

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“…Using an epithelial enrichment procedure (Sato et al, 2011), single cells were isolated with at least 85% viability (Leelatian et al, 2017). After additional viability enrichment (see STAR Methods) to >99% viability, ~1900 and ~700 colonic cells from two replicates were encapsulated and sequenced.…”

Section: Resultsmentioning

confidence: 99%

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Unsupervised Trajectory Analysis of Single-Cell RNA-Seq and Imaging Data Reveals Alternative Tuft Cell Origins in the Gut

et al. 2018

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Summary Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. However, computational tools that can infer developmental cell-state transitions reproducibly from such single-cell data are lacking. Here, we introduce p-Creode, an unsupervised algorithm that produces multi-branching graphs from single-cell data, compares graphs with differing topologies, and infers a statistically robust hierarchy of cell-state transitions that define developmental trajectories. We have applied p-Creode to mass cytometry, multiplex immunofluorescence, and single-cell RNA-seq data. As a test case, we validate cell state-transition trajectories predicted by p-Creode for intestinal tuft cells, a rare, chemosensory cell type. We clarify that tuft cells are specified outside of the Atoh1-dependent secretory lineage in the small intestine. However, p-Creode also predicts, and we confirm, that tuft cells arise from an alternative, Atoh1-driven developmental program in the colon. These studies introduce p-Creode as a reliable method for analyzing large datasets that depict branching transition trajectories. p-Creode is publicly available for download here: https://github.com/KenLauLab/pCreode.

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Section: Resultsmentioning

confidence: 99%

“…The epithelium was then dissociated into single cells with a collagenase/DNAse enzyme cocktail (2mg/ml Collagenase I, 2.5mg/ml DNAse1) in a modified protocol that maintains high cell viability (Leelatian et al, 2017). Cell viability was determined by counting Trypan Blue positive cells.…”

Section: Star Methodsmentioning

confidence: 99%

Unsupervised Trajectory Analysis of Single-Cell RNA-Seq and Imaging Data Reveals Alternative Tuft Cell Origins in the Gut

et al. 2018

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“…However, mass cytometry has just recently been developed and applied in solid tissues and organs (Diggins et al, in press ; Leelatian et al, 2016). One of the major limitations for flow cytometry is the need to generate a suspension of viable single cells derived from the tissue of interest.…”

Section: Introductionmentioning

confidence: 99%

“…(Codega et al, 2014; Pastrana et al, 2009; Rahman et al, 2015)). The protocol described here has been optimized to yield viable cells and to preserve known cell subsets from a variety of human tissues, including lymph nodes, gliomas, melanomas, and small cell lung cancer (SCLC) patient-derived xenografts (PDXs) (Leelatian et al, 2016). It is thus suitable for preparing single cells for fluorescence cytometry, mass cytometry, and other applications requiring isolated single cells.…”

Section: Introductionmentioning

confidence: 99%

“…It has been experimentally tested to preserve cell subsets detected using imaging platforms and maximize cell viability for cells from human tonsils, glioma tumors, melanoma tumors, and small cell lung cancer (SCLC) patient-derived xenografts (PDX) (Leelatian et al, 2016). Human tonsils, glioma tumors, and melanoma tumors were resected from patients and transported directly to the laboratory (within 1 hour after collection for human gliomas and melanomas, and within 4 hours after collection for human tonsils).…”

Section: Introductionmentioning

confidence: 99%

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Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

Leelatian

Doxie

Greenplate

et al. 2017

CP Molecular Biology

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Mass cytometry is a single cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >104 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single cell suspensions. This protocol establishes a primer for single cell analysis of solid tumors and tissues and has been tested with mass cytometry. The cells obtained from this protocol can be fixed for study, cryopreserved for long term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors.

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