Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.
BACKGROUND
We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell.


METHODS
A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence.


RESULTS
Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells.


CONCLUSIONS
This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.
Krylov
S N
SN
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.
Zhang
Z
Z
Chan
N W
NW
Arriaga
E
E
Palcic
M M
MM
Dovichi
N J
NJ
D001673:Biodegradation, Environmental; D002453:Cell Cycle / Q000502:physiology*; D004247:DNA / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D016195:G2 Phase; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D007364:Intercalating Agents; D007785:Lactose / Q000378:metabolism*; D008938:Mitosis; D012235:Rhodamines / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism
D005456:Fluorescent Dyes; D007364:Intercalating Agents; D012235:Rhodamines; D004247:DNA; D007785:Lactose