title: Manipulating ionic strength to improve single cell electrophoretic separations.
abstract: A capillary electrophoresis system with ultrasensitive two-color laser-induced fluorescence detection was used to probe the effect of ionic strength on single cell separations of glycosphingolipids. Differentiated PC12 cells were incubated with two ganglioside substrates tagged with different fluorophores within the BODIPY family such that two distinct metabolic patterns could be simultaneously monitored. Aspiration of single differentiated PC12 cells suspended in a phosphate-buffered saline solution showed excessive peak dispersion, poor resolution, and peak efficiencies below 100,000 theoretical plates. Aspiration of single differentiated PC12 cells suspended in deionized water corrected peak dispersion. Average peak efficiencies ranged between 400,000 and 600,000 theoretical plates. Improved performance was due to the dilution of the high salt concentrations inside of single neuronal-like cells to produce field amplified sample stacking. Single cell separations showed the highest resolution when aspiration of single differentiated PC12 cells suspended in deionized water were separated using a running buffer of high ionic strength. The improvement in resolution allowed for the identification of analytes not previously detected in single cell metabolism studies.
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Keithley
authors.forename: Mark P
authors.initials: MP
authors.lastname: Metzinger
authors.forename: Andrea M
authors.initials: AM
authors.lastname: Rosado
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Dovichi
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000737:chemistry; D002021:Buffers; D002454:Cell Differentiation; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism; D006028:Glycosphingolipids / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015394:Molecular Structure; D009994:Osmolar Concentration; D016716:PC12 Cells; D051381:Rats; D015203:Reproducibility of Results; D059010:Single-Cell Analysis / Q000379:methods*; D012965:Sodium Chloride / Q000737:chemistry; D014867:Water / Q000737:chemistry
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002021:Buffers; D005456:Fluorescent Dyes; D005732:Gangliosides; D006028:Glycosphingolipids; D014867:Water; D012965:Sodium Chloride