Flow Cytometry

A linear time-of-flight mass spectrometer was used as a detector for flow cytometry. These two techniques were coupled by a laser vaporization/ionization interface. The estimated mass detection limit of the combined system was 20 amol of serotonin standard with one laser pulse. An aqueous buffer at physiological pH was used to ensure compatibility with cells. Rat peritoneal mast cells (RPMCs) were dispensed into the mass spectrometer in a single file confined within a 20-micron-i.d. capillary. By using the mass spectrometer as a detector, no precolumn staining or derivatization is required. Determination of serotonin and histamine in individual cells was demonstrated. With this method, hundreds of cells can be analyzed within a few minutes. The average amounts of histamine and serotonin per RPMC were found to be 0.75 +/- 0.33 and 0.11 +/- 0.06 fmol, respectively. No correlation was found between the amounts of the two amines in each cell.

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Enhanced Inhibition of Human Anti-Gal Antibody Binding to Mammalian Cells by Synthetic α-Gal Epitope Polymers

Wang

Chen

Zhang

et al. 1999

J. Am. Chem. Soc.

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Neoglycopolymers of polyacrylamide backbone conjugated with varying densities of Galα1−3Galβ1−4Glcβ trisaccharide epitopes (α-Gal epitopes) were designed and synthesized to study the inhibition of the binding of human natural anti-Gal antibodies to either α-Gal-containing glycoproteins or α-Gal antigens on the surface of mammalian cells. An inhibition ELISA using mouse laminin and a flow cytometry assay using pig kidney cells (PK15) were established to determine the binding affinity of the synthesized polymers. In comparison to the α-Gal monomer (Galα1−3Galβ1−4GlcNHAcβ), the α-Gal polymers dramatically enhanced the inhibition of human anti-Gal antibodies (IgG, IgM, and IgA) binding to mouse laminin or mammalian cells. Increases of 7.8 × 103- and 5.0 × 104 -fold in inhibitory potential of polymer 7C to IgA and IgM (with IC50s of 7.0 and 5.6 nM respectively) were observed over the monomer in inhibition ELISA. The results also indicated that binding enhancement of α-Gal polymers is greater for anti-Gal IgA and IgM than for IgG. Such amplified binding differences among the three anti-Gal isotypes can be utilized to selectively inhibit or remove a particular isotype of anti-Gal antibodies. Moreover, it was demonstrated through the flow cytometry assay that certain α-Gal polymers are effective in inhibition of anti-Gal antibody (in human serum) binding to pig kidney (PK15) cells. Thus, such synthetic carbohydrate polymers may find practical applications in cell xenotransplantations.

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Flow Cytometry

Cited by 4 publications

References 79 publications

Structural Properties of Bacterial Inclusion Bodies

Structural Properties of Bacterial Inclusion Bodies

Direct Analysis of Single Rat Peritoneal Mast Cells with Laser Vaporization/Ionization Mass Spectrometry

Enhanced Inhibition of Human Anti-Gal Antibody Binding to Mammalian Cells by Synthetic α-Gal Epitope Polymers

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