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How to Maximize Immunofluorescence Multiplexing

Related Articles Chasing Rainbows Background glow Cutting crossover Unsticking Staying bright Combine dots and dyes to label the most proteins simultaneously. By doing this, Mario Roederer's group at the National Institute of Allergy and Infectious Diseases in Bethesda, Md., can image 17 colors in its flow cytometry experiments. Stuart Sealfon of Mount Sinai School of Medicine recommends saving quantum dots for the proteins that are the most difficult to pick up, while using org

By | December 1, 2007

Combine dots and dyes to label the most proteins simultaneously. By doing this, Mario Roederer's group at the National Institute of Allergy and Infectious Diseases in Bethesda, Md., can image 17 colors in its flow cytometry experiments. Stuart Sealfon of Mount Sinai School of Medicine recommends saving quantum dots for the proteins that are the most difficult to pick up, while using organic dyes for the rest. Combining also works well if you're looking at proteins that are present in very different quantities, says Jyoti Jaiswal of the Rockefeller University in New York City.

Keep numbers reasonable. With both quantum dots and organic dyes, each added fluorophore increases background fluorescence, and in general adds complexity. For that reason, Pok Man Mendy Chan of Mount Sinai School of Medicine limits her multiplex imaging to eight colors. Roederer's flow cytometry apparatus can handle 18 colors, he prefers to stick with 12 to 15 colors. "If you're doing a 12-color experiment on an 18-color machine with an 18-color palette to choose from, you have much more flexibility," he says. "It's much easier to develop the panels and the experiments."

Check toxicity. Although quantum dots are not necessarily toxic to cells, their preparation or coatings can make them toxic, Jaiswal says. This may not be a problem in fixed tissue, but it will be in live cells or organisms. The only way to know is to test any new preparations before use.

Compensate for blinking. For largely unknown reasons, quantum dots randomly flicker between bright and dark states. This is usually not a problem when labeling groups of proteins, but it "must be accounted for in single-particle tracking studies," says Diane Lidke of the University of New Mexico School of Medicine in Albuquerque. "There are several published algorithms for the blinking correction," she says. Some reports in the literature recommend adding reducing agents such as dithiothreitol or b-mercaptoethanol to suppress blinking, says Laurent Bentolila of the University of California, Los Angeles.

Use cell-penetration tricks. For intracellular imaging in live organisms, organic dyes are the only option; the size and hydrophobicity of quantum dots limit their cell penetration. For in vitro work, however, some tricks exist to circumvent that problem, says Jaiswal. Mixing quantum dots capped with negatively charged dihydroxylipoic acid into serum containing a transfection reagent allows delivery of quantum dots into the cytosol. Quantum dots also penetrate cells via carrier peptides or microinjections.

Be prepared to tweak. "These are all very hard techniques," Sealfon says, and adding colors increases difficulty. "There are a lot of different interactions that are not intuitive," says Roederer. "You lose relative sensitivity because you've got so many different things that you're measuring simultaneously." Reagents may perform differently with different dyes, and data analysis requirements also seriously increase with each color added. Roederer's group typically spends two months developing a 12- to 14-color panel for flow cytometry that they then use routinely. "It's not a simple system," he says. "But once you get it working, it's glorious!"

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