Repeatability of chemical cytometry: 2-DE analysis of single RAW 264.7 macrophage cells.
This report presents the use of 2-DE with ultrasensitive fluorescence detection as a chemical cytometry tool to characterize the protein and biogenic amine content of single cells from the RAW 264.7 murine macrophage cell line. Cells were sorted by cell cycle prior to 2-DE analysis. Cells in the G2/M phase of the cell cycle were aspirated into the first-dimensional capillary and lysed. The cellular contents were fluorescently labeled and first separated by capillary sieving electrophoresis (CSE). Over 380 fractions were transferred from the first-dimensional capillary to the second-dimensional capillary, where components were further separated by MEKC and detected by laser-induced fluorescence. Twenty-five spots common to the four electropherograms were fit with a 2-D Gaussian surface to determine spot position, width, and amplitude. The RSD in CSE mobility was 1.0 +/- 0.6%. The mean uncertainty in spot position was 1.3 times larger than the mean spot width in the CSE dimension. The average SD in MEKC migration time was 0.37 +/- 0.13 s, which is smaller than the average spot size in this dimension. Spot capacity was 200. The RSD in spot amplitude was 50%, reflecting a large cell-to-cell variation in component expression.
Sobhani
Kimia
K
Department of Chemistry, University of Washington, Seattle, WA 98195-1700, USA.
Fink
Susan L
SL
Cookson
Brad T
BT
Dovichi
Norman J
NJ
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008264:Macrophages / Q000166:cytology*; D051379:Mice; D015203:Reproducibility of Results