Premium
Repeatability of chemical cytometry: 2‐DE analysis of single RAW 264.7 macrophage cells
Author(s) -
Sobhani Kimia,
Fink Susan L.,
Cookson Brad T.,
Dovichi Norman J.
Publication year - 2007
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700017
Subject(s) - repeatability , cytometry , flow cytometry , macrophage , chemistry , biology , microbiology and biotechnology , chromatography , biochemistry , in vitro
Abstract 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.