Cell culture coverslips

TUNEL assays were performed per the manufacturer’s instructions (In Situ Cell Death Kit, TMR red; Roche Diagnostics, Indianapolis, IN). Sections incubated with DNase I (3000 U/ml in 50 mM Tris-HCl, pH 7.5, 1mg/ml BSA) for 10 minutes at 15–25°C were used as positive controls. To identify TUNEL+ cells, cryosections were first labeled with AlexaFluor (AF) 488-conjugated isolectin B4 (1:200; Molecular Probes, Euguene, OR), Iba-1 (1:100; ABCAM, Cambridge, MA), Thy-1 (1:100; ABCAM), or glutamine synthetase, and then with TUNEL. For TUNEL staining in cultured cells, cells were plated on cell culture coverslips (Thermoscientific, Rochester, NY). After staining, images were taken using a fluorescence microscope with five random images per section or coverslip. TUNEL-positive (+) cells were counted in each retinal section from lasered and non-lasered eyes injected with CCR3i vs. DMSO or PBS control or from laser-eyes treated with CCR3Ab vs. IgG control. In cultured cells, TUNEL+ cells determined by colabeling with DAPI stained nuclei were quantified and the mean of TUNEL+ cells in the five images from the same coverslip was used for comparison. There were at least 3–5 retinal cryosections or coverslips per condition.

For osteogenic and adipogenic differentiation, CBti MSCs at the end of P4 were seeded at a density of 4,000 cells/cm2 on cell culture coverslips (Thermo Fisher Scientific) and arranged in 24-well plates (Falcon®, Corning, Corning, NY, USA) in the presence of standard growth medium. At 70–80% cell confluence, the medium was replaced with specific differentiation media, then renewed every 3–4 days for 21 days. To induce adipogenic differentiation, cells were evaluated using the StemPro® Adipogenic Differentiation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The presence of intracellular lipid droplets was detected by standard staining with Oil Red O (Diapath, Bergamo, Italy). In parallel, cells were also grown using the StemPro® Osteogenic Differentiation Kit (Thermo Fisher Scientific). The presence of calcium deposits representing osteogenic differentiation were evaluated by von Kossa staining (Sigma-Aldrich). Cells were fixed with 10% formalin for 5 min at room temperature, incubated with 1% silver nitrate solution for 15 min and exposed to ultraviolet light for 2 h. Coverslips were rinsed with distilled water and 5% sodium thiosulfate to remove unreacted silver.

TUNEL staining was performed following manufacturer’s instructions (In Situ Cell Death Kit, TMR red; Roche Diagnostics, Indianapolis, IN) as described previously^{32 (link)}. Human RPE was plated on cell culture coverslips (Thermoscientific, Rochester, NY). After treatment, the cells were first fixed in 4% paraformaldehyde for 1 hour at room temperature. After three washes in PBS, cells were incubated with freshly prepared permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 mins on ice. After permeabilization, some cells were incubated with DNase I (3000 U/ml in 50 mM Tris-HCl, pH 7.5, 1 mg/ml BSA) for 10 minutes at 15–25 °C as positive controls. Cells incubated only with Label Solution without Enzyme Solution were used as negative controls. To identify TUNEL+ cells, cells were incubated with TUNEL reaction mixture (Label Solution and Enzyme Solution Mix in 10:1) for 60 mins at 37 °C in a humidified incubator in the dark. After two washes in PBS, cover slips were mounted with DAPI Fluoromount G. Images were taken under confocal microscope with five random images per coverslip. TUNEL+ cells determined by colabeling with DAPI stained nuclei were quantified, and the mean of TUNEL+ cells in the five images from the same coverslip was used for comparison. There were 5-6 coverslips per condition.