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The assessment of the viability of the cell culture is essential to determine whether the injected cells can recover after an experiment with the FluidFM technology. There is a large variety of assays which evaluate the viability of a cell culture; in our case, the cell damage is analyzed with a propidium iodide staining.
Propidium iodide (PI) is a fluorescent agent which is used as a DNA stain. PI cannot cross the membrane of living cells. However, in damaged or dead cells, which have compromised cell membranes, PI is able to pass through them and intercalates with the DNA in the nucleus. When the dye is bound to DNA, its fluorescence is enhanced, showing an excitation/emission maximum of 535/617nm. Therefore, the result of the assay can be easily imaged with fluorescence microscopy.
The protocol below describes the viability assessment of mammalian cells after an injection with Lucifer Yellow. A more detailed protocol can be found in the next file:
Mammalian cells injected with Lucifer Yellow solution (5 mg/ml in HEPES or TE buffer). Cells are still with the same medium used for the injection: a CO2 independent growth medium, supplemented with 2 nM L-Glutamine, 10% FBS and 1% penicillin/streptomycin.
5 mg/ml Propidium iodide stock solution (in PBS).
After the injection workflow is done, save the position of the injected cells.
5 minutes after the injection, add propidium iodide to the cell medium, at a final concentration of 2.5 µg/ml. Mix carefully without creating bubbles, nor moving the stage.
Image the cells after the staining with fluorescence microscopy, using the appropriate filters.
Report the number of viable cells: the nucleus of non-viable cells will be stained with propidium iodide.