Proteome profiler human cytokine array

Peripheral blood plasma collected under standard protocols, from one healthy donor, three low/intermediate-risk MDS patients, and six high-risk MDS patients, was stored at −80°C. Plasma from the same MDS patients as analyzed with CyTOF were used. Samples were assessed using the Proteome Profiler Human Cytokine Array (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions. Densitometry was performed using ImageJ (National Institutes of Health [NIH], Bethesda, MD).

Five-week-old astrocytes were washed in PBS and treated with 10 ng/mL recombinant human IL-1β (R&D) or PBS (vehicle) in astrocyte differentiation medium. Five hours after stimulation, 1 mL of medium was collected and flash frozen in liquid nitrogen for cytokine blot arrays. The remaining cells were detached as described above and counted to confirm a similar number across lines and experimental conditions. Three control (C1, C2, C3) and four BD astrocyte lines (BD1, BD2, BD4, BD6) were used for this experiment. The level of cytokines released in the medium was determined using Proteome Profiler Human Cytokine Array from R&D Systems according to the manufacturer's instructions. Comparative quantification of image intensity of cytokines was done using FIJI software (26). The intensity of cytokine dots (in stimulation group) was quantified using FIJI software and normalized per blot using positive controls provided by the manufacturer.