Method of Laser Irridation in Vitro Model
（Human RPE cell line ARPE-19）
We constructed a system in which one layer of densely cultured ARPE-19 cells was irradiated by a
diode laser with CW and MP mode. To avoid blockading of the diode laser, a phenol-red free cultured
medium was used. In order to uniformly provide the cell layer with laser energy, black paper was
spread out under the culture dish.
Expression of HSP70 protein after sub-lethal
Micropulse photocoagulation by
immnunocytochemistry
The up-regulation of HSP70 protein on sub-lethal micropulse laser photocoagulation site. At 3 hours (upper and lower left panels)
or 24 hours (right panels) after sub-lethal photocoagulation, the cell layers were immunostained with indicated antibodies and
counterstained with DAPI. Note that immunofluorescent signals of HSP70 expression were detected around laser irradiation sites
even in 24 hours (right panels).