Laurie J. Jones

Sensitive determination of cell number using the CyQUANT® cell proliferation assay

We describe here the development and characterization of the CyQUANT cell proliferation assay, a highly sensitive, fluorescence-based microplate assay for determining numbers of cultured cells. The assay employs CyQUANT GR dye, which produces a large fluorescence enhancement upon binding to cellular nucleic acids that can be measured using standard fluorescein excitation and emission wavelengths. The fluorescence emission of the dye-nucleic acid complexes correlated linearly with cell number over a large range using a wide variety of cell types. Under the recommended assay conditions, standard curves were linear (r(2)>0.995), detecting as few as 10-50 cells and as many as 25,000-50,000 cells with a single dye concentration, depending on cell type. Increasing the dye concentration extended the linear range of the assay to 100,000-250,000 cells. Results of cell proliferation and growth inhibition studies with the assay were similar to those obtained in published studies using other standard assays. CyQUANT assay measurements of serum-stimulated cell proliferation correlated well with measurements made using [3H]-thymidine. Also, the assay was used to analyze cellular DNA or RNA content, with the addition of a nuclease digestion step to the protocol. The assay procedure is simple and convenient, with no wash steps, and is readily amenable to automation.

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.

The fluorescent hydrazide, Pro‐Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff’s (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2–4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro‐Q Emerald 300 dye‐labeled gels and blots may be post‐stained with SYPRO Ruby dye, allowing sequential two‐color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid‐range UV illumination. Pro‐Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of α1‐acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro‐Q Emerald 300 dye. Besides glycoproteins, as little as 2–4 ng of lipopolysaccharide is detectable in gels using Pro‐Q Emerald 300 dye while 250–1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate‐polyacrylamide gels, two‐dimensional gels and on polyvinylidene difluoride membranes.

Simultaneous, two‐color fluorescence detection of total protein profiles and β‐glucuronidase activity in polyacrylamide gel

A dichromatic method for measuring the specific activity of β‐glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 β‐D‐glucuronide to detect β‐glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 β‐glucuronide is a precipitating substrate, it allows precise localization of β‐glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)‐polyacrylamide gel electrophoresis, carrier ampholyte‐mediated isoelectric focusing or two‐dimensional gel electrophoresis.

Simultaneous red/green dual fluorescence detection on electroblots using BODIPY TR-X succinimidyl ester and ELF 39 phosphate

A two‐color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY TR‐X dye to proteins immobilized on polyvinylidene difluoride membranes, followed by detection of target proteins using the fluorogenic, precipitating substrate ELF 39‐phosphate in combination with alkaline phosphatase conjugated reporter molecules. This results in all proteins in the profile being visualized as fluorescent red signal while those detected specifically with the alkaline phosphatase conjugate appear as fluorescent green signal. The dichromatic detection system is broadly compatible with ultraviolet epi‐ or trans‐illuminators combined with photographic or charge‐coupled device cameras, and xenon‐arc sources equipped with appropriate excitation/emission filters. The dichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots. Combining the detection approach with an Alexa Fluor 350 dye conjugated monoclonal antibody permits simultaneous fluorescence detection of two antigens and the total protein profile on the same electroblot.

Immunomagnetic cell separation, imaging, and analysis using Captivate ferrofluids

We have developed applications of CaptivateTM ferrofluids, paramagnetic particles (approximately 200 nm diameter), for isolating and analyzing cell populations in combination with fluorescence-based techniques. Using a microscope-mounted magnetic yoke and sample insertion chamber, fluorescent images of magnetically captured cells were obtained in culture media, buffer, or whole blood, while non-magnetically labeled cells sedimented to the bottom of the chamber. We combined this immunomagnetic cell separation and imaging technique with fluorescent staining, spectroscopy, and analysis to evaluate cell surface receptor-containing subpopulations, live/dead cell ratios, apoptotic/dead cell ratios, etc. The acquired images were analyzed using multi-color parameters, as produced by nucleic acid staining, esterase activity, or antibody labeling. In addition, the immunomagnetically separated cell fractions were assessed through microplate analysis using the CyQUANT Cell Proliferation Assay. These methods should provide an inexpensive alternative to some flow cytometric measurements. The binding capacities of the streptavidin- labled Captivate ferrofluid (SA-FF) particles were determined to be 8.8 nmol biotin/mg SA-FF, using biotin-4- fluorescein, and > 106 cells/mg SA-FF, using several cell types labeled with biotinylated probes. For goat anti- mouse IgG-labeled ferrofluids (GAM-FF), binding capacities were established to be approximately 0.2 - 7.5 nmol protein/mg GAM-FF using fluorescent conjugates of antibodies, protein G, and protein A.