Doctor of Philosophy, University of Cincinnati (2007)
Doctor of Medicine, University of Cincinnati (2005)
Garry Nolan, Postdoctoral Faculty Sponsor
Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.
View details for DOI 10.1038/nprot.2015.020
View details for PubMedID 25612231
Fluorescent cellular barcoding and mass-tag cellular barcoding are cytometric methods that enable high sample throughput, minimize inter-sample variation, and reduce reagent consumption. Previously employed barcoding protocols require that barcoding be performed after surface marker staining, complicating combining the technique with measurement of alcohol-sensitive surface epitopes. This report describes a method of barcoding fixed cells after a transient partial permeabilization with 0.02% saponin that results in efficient and consistent barcode staining with fluorescent or mass-tagged reagents while preserving surface marker staining. This approach simplifies barcoding protocols and allows direct comparison of surface marker staining of multiple samples without concern for variations in the antibody cocktail volume, antigen-antibody ratio, or machine sensitivity. Using this protocol, cellular barcoding can be used to reliably detect subtle differences in surface marker expression. © 2014 International Society for Advancement of Cytometry.
View details for DOI 10.1002/cyto.a.22573
View details for PubMedID 25274027
Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.
View details for DOI 10.1002/cyto.a.22075
View details for PubMedID 22693166