Propidium iodine solution

A viability assay was carried out using PI/FDA staining. First 20 μl PI (propidium iodine solution, 1 mg/ml, Sigma) and 10 μl FDA (fluorescein diacetate solution 1 mg/ml, Sigma) were added to ELS and incubated at room temperature for 90 s. Next the ELS were washed once in PBS (Invitrogen) and then florescence at 617 nm (excitation) and 520 nm (emission) measured, with 1 s and 150 ms exposure for PI and FDA staining respectively. The total FDA intensity was compared to the total PI plus FDA intensity using Nikon imaging software, giving both a cell membrane integrity and metabolic viability read-out.

A viability assay was carried out using PI/FDA staining. 20 μl PI (propidium iodine solution, 1 mg/ml, Sigma) and 10 μl FDA (fluorescein diacetate solution 1 mg/ml, Sigma) were added to ELS and incubated at room temperature for 90 s. The ELS were washed once in PBS (Invitrogen) and then florescence at 617 nm (excitation) and 520 nm (emission) measured, with 1 s and 150 ms exposure respectively. The total FDA intensity was compared to the total PI plus FDA intensity using Nikon imaging software, giving both a cell membrane integrity and metabolic viability read-out. This was carried out at 6, 24, 48, and 72 h post-thaw. The 6 h timepoint was chosen as this was the minimum time required to fully remove residual (pre-freeze) FDA-sensitive enzymes from non-viable cells.

The cells were fixed in 70% iced-cold ethanol and were incubated for 1 h at 4°C. The cells were washed, resuspended with 0.5 mg/ml RNase A (Sigma-Aldrich) for 1 h at 37°C and a 10 μg/ml propidium iodine solution (Sigma-Aldrich) was added in the dark at 4°C. The cells were observed by fluorescence microscopy and were analyzed using fluorescence-activated cell sorting (FACS).