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. Author manuscript; available in PMC: 2021 Aug 20.
Published in final edited form as: Amino Acids. 2020 Aug 20;52(8):1169–1180. doi: 10.1007/s00726-020-02881-w

Figure 2. LIVE/DEAD Cell assay.

Figure 2.

(a) Control for live cells (normal astrocytes without treatment) were incubated with a solution of EthD-1 (4 μM) and calcein-AM (2 μM) for 45 min at room temperature to determine the ratio of live cells (green) to dead cells (red) in typical astrocyte cultures (10X magnification was used for every image). Negative control astrocytes were incubated with 70% ethyl alcohol for 10 minutes at room temperature as a negative control for cell death. Then the cells were incubated with a solution of EthD-1 (4 μM) and calcein-AM (2 μM) for 45 minutes. The concentrations for DFMO, PTI and SPD were established from our previous dose response study (data not shown) and were DFMO (5 mM), PTI (30 μM) and SPD (10 μM). Astrocytes treated with DFMO, PTI, DFMO + PTI or SPD were incubated with a solution of EthD-1 (4 μM) and calcein-AM (2 μM) for 45 min at room temperature (n=18 in DFMO, PTI and DFMO+PTI and n=9 in SPD samples). (b) A summary of the percentage of dead cells in the various experimental groups. PTI (30 μM), DFMO (5 mM), PTI + DFMO and SPD (10 μM) do not cause significant cell death as compared to untreated astrocytes (denoted as live cells).