Then the fluorescence intensities on rMC-1 cells were measured with a fluorometer.
For the binding assay, rMC-1 cells were seeded in 96-well plates.
Retinal Müller-derived rMC-1 cells were chosen for the further mechanism study, as Müller cells in the retina are essential for the survival of the retinal neuronal cells, and rMC-1 also displayed the highest response for SERPINA3K.
The retinal Müller-derived rMC-1 cells exposed to H2O2 were stained with PI/Annexin-V, and necrotic and apoptotic cells were quantified by flow cytometry.
The control rMC-1 cells in the medium with 1% FBS showed only basal levels of green fluorescence, indicating a low intracellular Ca2+ concentration (Fig. 4B).
To identify the pathway involved in the H2O2-induced necrosis in rMC-1 cells, specific inhibitors for ERK, PI3K and PLC were used.
At the concentrations used to prevent the H2O2-induced rMC-1 cell death, both BAPTA and U73122 decreased the cell viability (Fig. S1).
We would like to thank Dr. Vijay Sarthy and Dr. Gail Seigel, who kindly provided us with the rMC-1 and R28 cell lines.
In contrast, FITC-BSA did not show any significant binding to rMC-1 cells in the same concentration range (Fig. 7A).
Müller-derived rMC-1 cells were treated with 400 µM H2O2 or 20 µM TBHP for 1, 2, 4 and 8 h in a medium containing 1% FBS.
Cultured rMC-1 cells were incubated with increasing concentrations (16 4096 nM) of FITC-labeled SERPINA3K for 1 h followed by 3 washes with PBS.
