24 well transwell plates

A healthy eye was removed from a 74-year-old patient undergoing orbital exenteration for an orbital tumour. The participant consented to his eye being utilised for this research. The eye was dissected on ice within an hour of exenteration, as previously described^{26 (link)}. Briefly, the eyes were dissected to remove the iris, the lens and the vitreous, followed by cutting the sclera into four quadrants. 4 mm diameter discs of neuroretina and RPE/choroid were trephined from the surrounding tissues using a Biopsy Punch (Fig. 5A). Both retinal and RPE/choroid explants were transferred to the inserts of a 24-well transwell plates (#353095, 0.4 μm pore size, Thermo Fisher Scientific) with culture medium of neurobasal-A with phenol red supplemented with 1% penicillin/streptomycin (#15140122; Gibco), 0.8 mM L-glutamine (#25030024, Thermo Fisher Scientific), 2% 50 × B27 supplement (#17504044, Thermo Fisher Scientific), and 1% 100 × N2 supplement (#17502048, Thermo Fisher Scientific). The volume of the medium was 400 µL in the insert and 900 µL in well. Neural retina and RPE/choroid explants were cultured in the same well for 24 h at 37 °C in 5% CO₂ incubator before AAV transduction (1 × 10¹¹ vg/well). Medium was changed every 2 days. Neuroretina and RPE/choroid explants were individually processed for immunostaining.

PBMCs were isolated from leukoreduction collars by Ficoll-Paque PLUS (17-1440-03, GE Healthcare, Pittsburgh, PA) density gradient centrifugation and cryopreserved prior to use. PBMCs were then thawed and plated in 24-well plates (2–2.5 millions/1 ml/well) pre-coated with anti-CD3 (2.5 μg/ml, HIT3a clone, Biolegend, San Diego, CA) in the presence of absence of soluble anti-CD28 (2 μg/ml, Cat. #302914, Biolegend) as well as adalimumab, etanercept, certolizumab pegol, and tocilizumab at indicated concentrations for 24 h before harvesting. In some experiments, Th (CD4+) cells were purified from resting PBMCs by using the human CD4+ T cell isolation kit (Cat. #130-045-101, Miltenyi Biotec, Bergisch, Gladbach, Germany) first before stimulation with anti-CD3 and anti-CD28. In the transwell experiments, Th cells were purified from 5 × 10⁶ of PBMCs and seeded on anti-CD3-coated inserts, whereas the remaining non-Th cells were plated in the bottom chambers of 24-well transwell plates (#140620, ThermoFisher, Waltham, MA). Exclusion of non-Th subsets from PBMCs was carried out with CD8 (#130-045-201, Miltenyi), CD19 (#130-050-301, Miltenyi), or CD14 (#130-050-201, Miltenyi) MicroBeads.