Cryogenic tubes

Blood serum samples were collected from BLSA participants (AD = 26, CN = 20) at time of AD diagnosis, 43 of whom were included in the autopsy sample, at the NIA Clinical Research Unit in Harbor Hospital, Baltimore. Details on collection and processing have been published previously^{55 (link)}. Briefly, venous blood samples were collected between 6 and 7 a.m. following an overnight fast. Serum samples were aliquoted into 0.5-mL volume in Nunc cryogenic tubes and stored at −80 °C until further use. Samples were not subject to any freeze–thaw cycles prior to proteomic assays. Additional details on sample selection have been published previously^{55 (link)}. ROS participants (AD = 29, CN = 22) provided blood serum at the time of AD diagnosis, all of whom were included in the autopsy sample, as described previously⁵⁶ .

Fresh tumor tissue was obtained from the resection of a metastatic lesion to the dermis to establish a new cell model (USZ20-ESOS1). The tumor tissue was mechanically dissected into small pieces and treated with liberase™ (TM Research Grade, Merck) at a concentration of 1 mg/ml for 4 h. Cells were plated in 6 well ultra-low attachment plates (ULA; Corning) and maintained in sarcoma culture media (Supplementary Table 1). Sarco-spheres were passaged every 2–2.5 weeks by using Tryple-LE (Gibco) for 20 min in a water bath at 37 °C and transferred to a fresh tissue culture plate. Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Furthermore, cells were able to attach as a monolayer and can be grown in 2D when kept on collagen-coated flasks (Thermo-Fisher). Cells were cryo-preserved in cryogenic tubes (Nunc) in our biobank. For freezing, the sarco-spheres were digested with Tryple-LE (Gibco) and frozen down in heat-inactivated horse serum (Gibbco) supplemented with 10% DMSO (Sigma) in a cell freezing container at -80°C for 3–6 days prior transferring the cells to liquid nitrogen. For defrosting the cells, warm cell culture media was added to the cryogenic tube (Nunc). Cells were washed and spun down twice and then placed in ultra-low attachment plates with fresh media.