Z lehd fmk

After the aggregates were transferred to adhesion plates, the culture medium was changed to Neurobasal Medium (NM, Gibco, Life Technologies Corporation, Grand Island, NY, USA), containing 0.5 mM of L-Glutamine (Gibco) and 2% NeuroBlew-21 (MACS) on D26. Q-VD-Oph (10 µM; Abcam, Cambridge, UK), Necrox-5 (30 µM; Enzo, Farmingdale, NY, USA), and Z-LEHD-FMK (10 µM; Medical & Biological Laboratories, Tokyo, Japan) were added to the respective plates on D26. On D27, the plates were transferred and sealed in a container with AnaeroPack Kenki (Mitsubishi Gas Chemical, Tokyo, Japan), an oxygen absorber/carbon dioxide generator, to simulate hypoxic injury at 37 °C for 18 h. The plates were then removed from the container and incubated at 37 °C for 4 h under normal atmospheric conditions to simulate reoxygenation. The duration of hypoxia and reperfusion was deemed appropriate based on a previous report [19 (link)], and we further conducted multiple experiments in order to adjust the conditions under which both cell death and their rescue by the drug could be observed.