Dmi8 s inverted microscope system

Spinal cord sections were stained with DAPI (F6057, Sigma Aldrich) and imaged at 20x (Leica Dmi8-S Inverted Microscope System). All labeled neurons were hand-counted, and count accuracy was assessed by comparison with 15 randomly selected slides counted by a second blinded observer. Student’s t-test showed no significant differences between counts obtained by different observers. Accurate estimations of individual labeled neuron count were calculated using Abercrombie’s correction to account for duplicate counting of individual motor neuron nuclei.^{[30 (link)]}

Lactate dehydrogenase (LDH) is an enzyme released upon cell breakdown, and therefore an aggregate measure of cell death. Measurements of LDH levels in samples of cell media used a commercially available colorimetric assay (CytoTox 96® Non-Radioactive Cytotoxicity Assay, Promega, Madison, WI). Identical culture media from wells without cells assayed alongside the experimental groups served as a control.
Live-cell imaging occurred after 24 hours in culture, with fluorescent cell markers to identify cell survival status. Calcein AM (Thermo Fisher Scientific, Waltham, MA) labeled living cells, permeating the intact cell membrane and undergoing conversion to its fluorescent form via intracellular esterase activity. Propidium Iodide (Thermo Fisher Scientific), which is membrane impermeant, labeled dead cells with degraded cell membranes. Hoechst (Thermo Fisher Scientific) labeled DNA in all cells regardless of cell survival status. 20x images were obtained from the center of each well using an inverted microscope (Leica Dmi8-S Inverted Microscope System), followed by blinded quantification of cell survival in ImageJ.^{[28 ]}