Luminex platform

Mouse serum samples were assayed for mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgE, and IgM using ProcartaPlex Mouse Antibody Isotyping Panel on the Luminex platform (Affymetrix, Santa Clara, CA, USA), according to manufacturer’s instructions. In brief, samples were thawed on ice. Beads were mixed and washed and subsequently incubated overnight at 4°C with standards or with 1:500 or 1:50,000 diluted samples. After washing, the beads were incubated with detection antibody mix for 30 min at room temperature. The beads were washed and incubated for 30 min at room temperature with streptavidin-PE. After washing the beads were measured with a Luminex instrument (Bio-Plex 200, Bio-Rad), which was calibrated using Bio-Rad calibration beads. Standard curves were calculated using 5-parameter logistic regression in Bio-plex 5.0 software.

Cytokine responses in BALF samples from NHPs were examined using the cytokine monkey magnetic 29-plex panel for the Luminex platform (Thermo Fisher Scientific). Cytokines that were analyzed included G-CSF, IFN-γ, IL-10, IL-12, IL-17A, IL-2, IL-4, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, EGF, eotaxin, FGF-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), HGF, IL-1β, IL-1RA, IL-15, IL-5, IP-10, I-TAC, MDC, MIF, MIG, and VEGF-A. The procedure was performed according to the manufacturer’s recommendations; BALF samples were tested undiluted, and test plates were run using a Luminex Magpix instrument.

For sample preparation, LUHMES cells were lysed in Laemmli buffer and boiled at 95 °C for 5 min. The lysate was centrifuged for 1 min at 10,000× g through NucleoSpin Filters (Macherey-Nagel, Düren, Germany) to remove nucleic acids. The DigiWest method was used exactly as described earlier [31 (link)]. Briefly, the analysis procedure started with an electrophoretic separation and blotting of proteins onto nitrocellulose membranes. Then, proteins were biotinylated and eluted from small pieces of the membrane (each lane was cut into 96 evenly large strips). Eluted proteins were incubated with color-coded streptavidin-coated Luminex beads (Luminex FlexMAP 3D system). Beads were incubated with primary antibodies for the selected proteins (acetylated tubulin; phosphorylated (at serine-51) and non-phosphorylated eIF2-alpha) and a phycoerythrin-labelled secondary antibody (all reagents and devices by the Thermo Fisher LUMINEX platform (Waltham, MA, USA)).

Cytokines released by primary microglia in the culture medium were quantified by ELISA using the following commercial kits according to the manufacturer’s instruction: Mouse TNF alpha Uncoated ELISA kit (88-7324-22, Invitrogen), Rat IL-6 DuoSet ELISA (DY506) and Rat IL-1β DuoSet ELISA (DY501) (R&D Systems), and Human IL-1β DuoSet ELISA (DY201, R&D Systems). Cytokines and growth factors levels were normalized to total protein content in the corresponding cell lysates. For the quantification of cytokines in brain homogenates (TNF, IL-6), aliquots of brain homogenates containing equivalent amounts of proteins were analysed using a Luminex platform (Thermo Fisher Scientific) by Eve Technologies Corporation (Calgary, AB, Canada).