Endothelial C/EBPβ/AEP Pathway Drives Vascular Deterioration and Systemic Aging
Endothelial C/EBPβ/AEP Pathway Drives Vascular Deterioration and Systemic Aging
Mice were euthanized, and quadriceps femoris, brain, and liver were dissected. Magnetic‑activated cell sorting (MACS) of CD31+ endothelial cells: Cells were centrifuged at 400g for 5 min, and the pellet was resuspended in 80 µL of buffer (PBS with 0.5% BSA and 2 mM EDTA), followed by addition of 20 µL of CD45 microbeads. After a 15‑minute incubation at 4°C in the dark, cells were centrifuged at 300g for 10 min. The cell suspension was loaded onto an LS column pre‑rinsed with 1 mL of buffer. The CD45‑negative flow‑through was collected, and the column was washed with 2 mL of buffer. The CD45‑negative fraction was centrifuged at 400g for 5 min, resuspended in 90 µL of buffer with 10 µL of CD31 microbeads, and incubated for 15 min at 4°C in the dark. After centrifugation at 300g for 10 min, cells were resuspended in 1 mL of buffer and applied to a fresh LS column. The column was washed with 2 mL of buffer, then removed from the magnet. CD31‑positive endothelial cells were eluted by flushing with 4 mL of buffer using the plunger. The eluted cells were centrifuged at 400g for 5 min and resuspended in RPMI‑1640 for downstream applications. After MACS sorting, purified CD31+ endothelial cells were collected by centrifugation. Cell pellets were lysed using the proprietary lysis buffer provided in the Chromium Single Cell 3' v3.1 kit (10X Genomics). RNA was released from the lysed cells and subsequently reverse-transcribed into cDNA within the Gel Bead-in-Emulsions (GEMs). The resulting barcoded cDNA was then purified and amplified for downstream library construction. Sequencing libraries were constructed using the Chromium Single Cell 3' v3.1 reagent kit (10X Genomics).