Cell viability assessment


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:


MATERIALS

  • 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).

PROCEDURE

  1. After the injection workflow is done, save the position of the injected cells. 

  2. 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.

  3. Image the cells after the staining with fluorescence microscopy, using the appropriate filters. 

  4. Report the number of viable cells: the nucleus of non-viable cells will be stained with propidium iodide. 


Example of cell viability assay on HeLa cells after the injection with Lucifer Yellow. Cell injection was first checked with the observation with fluorescence microscopy (A). Cell viability was then assessed by PI staining: cells were observed with bright field (B) and fluorescence (C) microscopy. As observed, none of the injected cells was stained with PI. 


Troubleshooting

Harmful injection conditions - mechanical stress

When the selected injection parameters are too harmful for the cells, they cannot recover after the injection. As an example, in the picture below, HeLa cells were injected with a setpoint of 800 mV, with an approach speed of 100 um/s, with a pressure of 100 mbar and an injection time of 10 s. Cell viability was assessed with propidium iodide staining after 4 h of injection: around 35% of the injected cells were stained with PI, indicating that the chosen conditions for injection were too harmful for some cells.