Rapid Sampling for Single‐Cell Analysis by Capillary Electrophoresis

Nelson, Alan; Allbritton, Nancy L.; Sims, Christopher E.

doi:10.1016/s0091-679x(06)82026-1

Cited by 11 publications

(9 citation statements)

References 25 publications

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“…Pretreatment of cells, if any, was performed by removing media from a cell chamber, rinsing twice with 37 °C serum-free DMEM and then adding the appropriate inhibitor dissolved in serum-free DMEM and incubating at 37 °C for 10–20 min. After pretreatment, a cell chamber was placed on the microscope stage of the customized single-cell CE system, 20 a cell was visualized and microinjected with a mixture of peptide reporter and internal standard using a Transjector 5246 microinjection system (Eppendorf AG, Hamburg, Germany). The cell chamber temperature was maintained at 37 °C using a constant flow of warmed extracellular buffer (ECB; 10 mM HEPES, 135 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , pH 7.4) during microinjection, reporter incubation, and cell lysis.…”

Section: Methodsmentioning

confidence: 99%

Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

et al. 2013

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A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens.

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Section: Methodsmentioning

confidence: 99%

Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

et al. 2013

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“…38 The cell chamber was precoated with Cell-Tak (7.7 μg/mL) to enhance adhesion of the cells to the glass coverslip that formed the base of the chamber. Cell chambers were placed on an inverted microscope and imaged using an oil immersion 100× objective.…”

Section: Ce Analysis Of Intact Cellsmentioning

confidence: 99%

“…38 Fused-silica capillaries (50 μm i.d., 360 μm o.d., Polymicro Technologies, Phoenix, AZ) were used for the analyte separations. The capillary length was 18 cm from the inlet to the optical window with a total length of 41 cm.…”

Section: Capillary Electrophoresismentioning

confidence: 99%

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

et al. 2008

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Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...

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“…Cells were maintained at 37 °C in a humidified 5% CO 2 atmosphere. Cells for use in single-cell, CE experiments were plated the day before the experiment in custom-made cell chambers created from a #1 glass coverslip to which a silicon “O” ring was attached using poly(dimethyl siloxane) (Sylgard 184) 74. For experiments using serum-starved cells, the media was exchanged with DMEM lacking FBS 8-12 h before the start of the experiment.…”

Section: Methodsmentioning

confidence: 99%

Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

2011

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SummaryA single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (Bodipy Fl PIP 2 ) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomoles. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that Bodipy Fl PIP 2 was converted to phosphatidyl-inositol 3,4,5 trisphosphate (Bodipy Fl PIP 3 ). Bodipy Fl PIP 3 could be back converted to Bodipy Fl PIP 2 by the phosphatase PTEN. When Bodipy Fl PIP 2 was added to a cell lysate, 1.4 fmoles of the Bodipy Fl PIP 3 were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of Bodipy Fl PIP 3 to a cell lysate yielded 3 fmoles of Bodipy Fl PIP 2 per ng of protein in 8 min. Both Bodipy Fl PIP 2 and Bodipy Fl PIP 3 were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the FcεR1 receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of Bodipy Fl PIP 3 generated per cell increased 4-fold over that of unstimulated cells. This production of Bodipy Fl PIP 3 was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level.

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Rapid Sampling for Single‐Cell Analysis by Capillary Electrophoresis

Cited by 11 publications

References 25 publications

Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

Single-cell analysis of phosphoinositide 3-kinase and phosphatase and tensin homolog activation

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