Human magnetic luminex assay lxsahm

Blood samples were collected at 8:00 a.m. from participants who had fasted overnight. The BDNF and CNTN1 levels in plasma samples were obtained using the Human Magnetic Luminex Assay (LXSAHM; R&D Systems, Minneapolis, MN, USA). The assays were conducted according to the protocol that was provided by R&D Systems. Briefly, a 96-well filter-bottom microplate (Millipore, Billerica, MA, USA) was blocked for 10 min with assay buffer (R&D Systems). To generate standard curves, four-fold serial dilutions of standards were prepared in serum diluent (R&D Systems). 50 μL of standards and plasma samples (1:2 dilution) were added to the wells, which contained 50 μL of the immunobead mixture. The microplate was incubated for 40 min at room temperature in the dark and then washed three times with washing buffer (R&D Systems) using a vacuum manifold. A mixture of biotin-conjugated secondary antibodies (R&D Systems) was added. After 40 min of incubation and three washes, streptavidin-phycoerythrin (R&D Systems) was added. After 10 min, the immunobeads were washed three times, resuspended in 50 μL assay buffer, and analyzed using the Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA, USA).

Cell culture supernatants were analyzed to measure IL production by PBMCs. The whole content of each well was collected in a microcentrifuge tube, centrifuged at 400 g for 5 min and the supernatant recovered and stored in a new tube.
The concentration of IL-1β, IL-2, IL-6, IL-10 and TNF-α were measured with Milliplex MAP Human Cytokine/Chemokine Multiplex Immunoassay (Millipore, Merck) and Human Magnetic Luminex Assay LXSAHM (R&D systems) following manufacturer´s instructions. RPMI culture medium was applied as background. A MAGPIX device with xPONENT software was used for fluorescence measurement and median fluorescent intensity data were analyzed using 5-parameter logistic method for calculating cytokine concentrations.