C576065

hRECs (PB-CH-160-8511, Pelo Bioscience) were maintained in EGM-2 MV medium (CC-3202, Lonza) under treatment conditions for 2 weeks. In addition to the 2 treatment conditions with 1000 U IFN-β (8499-IF-010, R&D Systems) and 33 μg/mL cGAMP (tlrl-nacga23m, InvivoGen), a control arm was performed. Meanwhile, the cells underwent 3 passages (p7–p9) and the medium was changed every other or every third day. After the last passaging step, cells were split into a 96-well plate for the readout assay.
Primary mRECs (C576065, Cell Biologics) were cultured in Complete Mouse Endothelial Cell Medium (M1168, Cell Biologics) at 37°C in an incubator containing 5% CO₂. The medium was changed every other day until the cells were confluent for use. For the flow cytometry cell senescence assay and the ELISA experiment, mRECs were seeded in a 6-well plate at a concentration of 0.2 million cells per well. For the confocal imaging cell senescence assay, mRECs were seeded on a cell culture cover glass (NC0620709, Thermo Fisher Scientific) and laid on the bottom of a 24-well plate (3527, Corning) at 0.1 million cells per well. After 24 hours of incubation, mRECs were treated with 0.1% DMSO (control), cGAMP (20 μg/mL), STING inhibitor (2 μg/mL; inh-h151, Invivogen), or IFN-β (1000 U/mL; 12400-1, R&D Systems) for an additional 1, 2, or 4 days, respectively.

Bone marrow-derived macrophages (BMDMs) were collected as described previously [22 (link)]. Briefly, bone marrow cells were flushed from the tibiae and femurs of C57BL/6 mice and seeded at 1 × 10⁶ cells/well in 24-well culture plates in DMEM supplemented with 10% FBS, L-glutamine (2 mM), 1% penicillin/streptomycin, and 15% L929-conditioned medium (LCM). Cells were incubated at 37°C in a humidified 5% CO₂ incubator. On day 7, adherent macrophages were gently scraped, and all the cells were resuspended, centrifuged, and seeded at 2 × 10⁶ cells/well in 6-well culture plates in culture medium for 24 h. The purity of the BMDMs was routinely >95%, as confirmed by integrin alpha M (ITGAM; CD11b) staining (Cell Signaling Technology; CAT No. 17800). For glutamine deprivation, cells were cultured in glutamine-free and pyruvate-free DMEM with 4.5 g/l glucose (Thermo Fisher Scientific; CAT No. SH30081) and 10% dialyzed FBS (Thermo Fisher Scientific; CAT No. 30067334).
Mouse retinal microvascular endothelial cells (MRMECs) were purchased from Cell Biologics (CAT No. C57-6065). The cells were cultured in complete mouse endothelial cell medium (ECM) mixed with 1% penicillin and streptomycin antibiotics (Cell Biologics; CAT No. M1168) under 5% CO₂ at 37°C. Cells from passages 3 to 7 were used for experiments.