96 well opaque plate

Caspases 3 and 7 activity were measured in RGCs by using a luminescent assay kit (G8091; Promega, Madison, WI, USA). Purified RGCs were seeded (~10,000 RGCs/well) in 96-well opaque plate (BD Falcon, Franklin Lakes, NJ, USA). After the appropriate treatments, 50 lL of Caspase-Glo 3/7 assay reagent were added into each well, shaken for 30 seconds on an orbital shaker, and incubated for 1 hour at room temperature. The total volumes of 100 lL of each well were then transferred into a white-walled 96-well plate (BD Falcon). Luciferase signal was then read by a luminescent plate reader (Infinite m200; Tecan Group Ltd., Männedorf, Switzerland).

Cell proliferation study was performed by a fluorimetric dsDNA quantification kit (Quant-iT™, PicoGreen ®, Molecular Probes, Invitrogen, USA). The specimens for 1, 3 and 7 days of culture were washed twice with PBS and transferred into Eppendorf tubes containing 1 ml of ultra-pure water. The samples were left to incubate for 1 h at 37 °C and 5% CO 2 humidified atmosphere and then frozen at -80 °C until further analyses. For the DNA quantification, the samples were thawed and sonicated for 20 min. The DNA standards were prepared at concentrations 0 μl.ml -1 , 0.2 μl.ml -1 , 0.5 μl.ml -1 , 1 μl.ml -1 and 1.5 μl.ml -1 . The PicoGreen, TE buffer and the sample were added at a 96-well opaque plate (Falcon). The fluorescence was measured in a microplate reader (Synergie HT, Bio-TEK, USA) using an excitation wavelength of 480 nm and an emission wavelength of 528 nm. For each sample, the DNA concentration was calculated using a standard curve that relates DNA concentration with fluorescence intensity. The results were normalized by the specific area.

A cell proliferation assay was performed using a double stranded DNA (dsDNA) quantification kit (Picogreen®, Invitrogen). Thus, HFF-1 (3000 cells/cm 2 ) and HaCaT cells (3000 cells/cm 2 ) were seeded separately on matrices of randomly aligned ELR-click fibers deposited on glass coverslips, which were placed in 24-well plates and subsequently treated with 1% BSA to avoid unspecific cell adhesion to the carrier. Next, cells were incubated for 4 h (adhesion test), and 1, 3 and 7 days (proliferation test). After culture, samples were washed twice with DPBS and 250 µL of ultra-pure water. The samples were frozen at 193 K until analysis. For DNA quantification, the samples were thawed and the freeze-thaw cycle was repeated twice to achieve cell lysis. The reagent, TE buffer and samples were added in triplicate to a 96-well opaque plate (Falcon). Fluorescence was measured using a microplate reader, with an excitation wavelength of 480 nm and an emission wavelength of 528 nm.