OL cell death in culture was determined using the LDH assay (Takara Bio, Inc., Madison, WI). Briefly, 100 µl of tissue culture media from each experimental group was added to a 96 well plate and 100 µl of LDH reagent was added to each well. Plates were incubated for 30 min at 25°C and absorbances were read on a microplate reader at 548 nm wavelength. The media from the HUCBC only wells served as a control of HUCBC death. The absorbance of HUCBC only media, as well as the absorbance of media only, was subtracted from the total absorbance of the OL wells to eliminate background LDH activity.
A standard curve was generated from experiments using known concentrations of OLs. In these experiments, the cells were lysed with 2% Triton X-100 and the amount of LDH release was quantified. The absorbances derived from the known concentrations were used to produce the standard curve. The reported OL cell numbers from the hypoxia experiments were extrapolated from this standard curve.
A standard curve was generated from experiments using known concentrations of OLs. In these experiments, the cells were lysed with 2% Triton X-100 and the amount of LDH release was quantified. The absorbances derived from the known concentrations were used to produce the standard curve. The reported OL cell numbers from the hypoxia experiments were extrapolated from this standard curve.
