Both extracellular and intracellular BDNF protein levels were measured at 3, 6, 12, 24, and 48 hours after ES by quantitative sandwich enzyme immunoassay (Chemicon International, Temecula CA) according to the manufacturer’s instructions. This technique can measure both human and rat BDNF. The culture medium and the cell lysate were collected as the samples for extracellular and intracellular BDNF, respectively. To lyse the Müller cells, the cells were soaked in 200 μL of lysis buffer (RIPA buffer; Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Protease Inhibitors Mixture for Protease and Esterase; Wako, Osaka, Japan). The lysate was centrifuged at 15,000 rpm for 10 minutes at 4°C, and the BDNF in the supernatant was measured as intracellular BDNF.
The optical density of each sample was measured at 450 nm with the correction wavelength set at 540 nm (ARVO
MX; PerkinElmer Japan, Kanagawa, Japan). Each measurement was performed in duplicate in six independent runs. The absolute protein amount of BDNF was calculated by the following equations:
\[\mathrm{Extracellular\ BDNF}\ (\mathrm{pg}){=}\mathrm{medium\ concentration}\ (\mathrm{pg}/\mathrm{mL}){\times}2\ (\mathrm{mL},\ \mathrm{medium\ volume})\]
,
\[\mathrm{Intracellular\ BDNF}\ (\mathrm{pg}){=}\mathrm{lysate\ concentration}\ (\mathrm{pg}/\mathrm{mL}){\times}0.2\ (\mathrm{mL},\ \mathrm{lysate\ volume})\]
, and
\[\mathrm{Total\ BDNF}\ (\mathrm{pg}){=}\mathrm{sum\ of\ extra-\ and\ intracellular\ BDNF}\ (\mathrm{pg}).\]
The expression of the protein BDNF in the ES group was compared with that in the control group. The relative data are presented as the mean multiple of change ± SD calculated from six separate experiments. The Wilcoxon signed rank test was used to analyze the statistical significance (P < 0.05) of differences between the medians.