Live Cell Labeling Protocol

Live cell labeling refers to the process of labeling cells with specific biomolecules, such as fluorescent dyes, antibodies, or other probes, while the cells are still alive and active. This technique is commonly used in cell biology, immunology, and microscopy applications to track and visualize cellular structures, processes, or specific molecules within living cells.

• Prepare the labeling buffer for the specific application and cell type.
• Harvest the cells and wash them with PBS to remove any residual media and serum. Centrifuge the cells at a suitable speed and duration to pellet them.
• Resuspend the cell pellet in the labeling buffer at a suitable concentration for the application. Be sure to handle the cells gently to ensure their viability.
• Add the fluorescent dye to the cell suspension. Incubate for 15 minutes in a 37°C + CO₂ cell culture incubator. Protect the samples from light during this step, if required by the specific dye.
• Gently replace the ligand-containing medium with an equal (or greater) volume of warm fresh medium (or 1 × PBS [pH 7.5]). Repeat this two times ending with warm complete medium, for a total of three complete rinses.
• Incubate cells in a complete culture medium at 37°C + CO₂) in a cell culture incubator for 30 minutes to wash out unbound ligands.
• Replace the medium with an equal volume of fresh warm culture medium (use of medium lacking phenol red may improve imaging).
• Transfer to a microscope and capture images.

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• To improve long exposures, it is recommended to use longer wavelength fluorescent reagents. These reagents require lower excitation power, have lower phototoxicity, and provide a healthier cellular state.
• It is important to optimize staining for specific assay readings, spectral compatibility, and signal-to-noise ratio.
• To reduce background fluorescence, remove the labeling solution and rinse with freshly prepared media.
• Multiple labeling is achieved using multiple fluorophores. It is recommended to use a fluorescence spectrum viewer to ensure minimal spectral overlap between fluorescent dyes.

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