Single-cell mass cytometry adapted to measurements of the cell cycle.
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.
Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Gregory K
GK
Behbehani
Sean C
SC
Bendall
Matthew R
MR
Clutter
Wendy J
WJ
Fantl
Garry P
GP
Nolan
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001854:Bone Marrow Cells / Q000378:metabolism / Q000502:physiology; D059447:Cell Cycle Checkpoints; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002469:Cell Separation; D019925:Cyclin A / Q000378:metabolism; D056744:Cyclin B1 / Q000378:metabolism; D004261:DNA Replication; D005434:Flow Cytometry; D006410:Hematopoiesis; D006657:Histones / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016160:Retinoblastoma Protein / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000378:metabolism / Q000502:physiology; D028561:Transition Elements / Q000737:chemistry
D000906:Antibodies; C534699:CCNB1 protein, human; D019925:Cyclin A; D056744:Cyclin B1; D006657:Histones; D008565:Membrane Proteins; D016160:Retinoblastoma Protein; D028561:Transition Elements