Bio plex manager software v 6

Brain homogenates were centrifuged at 10,000 × g for 10 min at 4 °C. A volume of 50 μL of supernatant was taken for the determination of cytokine and chemokine levels using a commercial multiplex mouse cytokine magnetic bead–based immunoassay (Bio-Plex Pro Mouse Cytokine 23-plex Assay; Bio-Rad Laboratories) according to the manufacturer’s instructions. Mean fluorescence intensity from all the bead combinations was analyzed using of the Bio-Plex system equipped with the Bio-Plex Manager Software v6.0 (Bio-Rad Laboratories).

Concentrations of plasma cytokines were measured using the Bio-Plex Pro^™ Human Cytokine 17-plex Assay kit (Bio-Rad Laboratories Inc, California, USA). Reconstituted standards, cytokine-specific coupled beads, detection antibodies and 50 µl of thawed serum samples were combined according to manufacturer’s instructions and data acquired with the Bio-Plex^™ 200 reader (Bio-Rad Laboratories Inc, California, USA). Data were analysed using Bio-Plex Manager™ software v6.0 (Bio-Rad Laboratories Inc, California, USA).

GCF levels of ten inflammatory mediators were assayed using the multiplex assay technique (ProcartaPlex multiplex immunoassays human Th1/Th2 cytokine panel; Affymetrix eBioscience, Santa Clara, CA, USA) according to the manufacturers' instruction manual. The assay was read using a Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA) with the Bio-Plex Manager software v6.0 (Bio-Rad) [20 (link)]. To demonstrate a high level of correlation between measurements, duplicate measurements were performed with a subset of samples, for which the intraclass correlation coefficients varied from 0.95 to 1.0 (P < 0.001). The following cytokines were measured: interleukin- (IL-) 1β, IL-2, IL-4, IL-5, IL-6, IL-12p70, IL-13, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF).

Brain homogenates (350 μL) of mice sacrificed prior to (day 0) and on days 6, 8 and 10 following the infection were centrifuged at 13,000 × g for 10 min at 4°C. A volume of 100 μL of the supernatant was used for cytokine and chemokine measurements using the Bio-Rad Bio-Plex mouse cytokine group I plex assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. Results were analyzed with the Bio-Plex system equipped with the Bio-Plex Manager Software v6.0 (Bio-Rad Laboratories).

Brain cortices were homogenized and lysed using a Bio-Plex cell lysis kit (BioRad, 171-304011), with the addition of protease inhibitor cocktail (Thermo Scientific Pierce, Spain) following the manufacturer’s directions. Lysates were centrifuged at 14,500 rpm and 4 °C for 12 min. Supernatants were stored at − 80 °C until used. Protein content was determined by Bradford assay [33 ]. Samples were normalized to 7.5 μg/μl in 0.5% bovine serum solution, and 50 μL of each sample was added to the Bio-Plex kit. Cytokine protein was quantified using the Bio-Plex Pro™ Luminex Cytokine panel (BioRad 10,014,905) and read out using a Bio-Plex Manager Software v 6.0 and Bio-Plex 200 system (Bio-Rad, Spain). Data were expressed in pg/mg total protein, by Bradford analysis [33 ]. G-CSF was excluded from analysis because it was not detectable above background.

The concentration of the BMP family in the supernatant of ALX1^165F/165F NCC was measured using a bead‐based multiplex array (Forsyth Institute, Cambridge, MA). Manufacturers’ protocols were followed for all panels. Reagents were prepared as per kit instructions. Assay plates (96‐well) were loaded with assay buffer, standards, supernatant from the ALX1^165F/165F NCC, and beads and then covered and incubated on a plate shaker (500 rpm) overnight at 4°C. After primary incubation, plates were washed twice. Following this, the detection antibody cocktail, consisting of BMP‐specific antibody with biotin (1:10) and the detection antibody streptavidin conjugated with PE (1:25), was added to all wells; the plates were covered and left to incubate at room temperature for 1 h on a plate shaker. After the incubation, streptavidin‐phycoerythrin fluorescent reporter was added to all wells, and the plate was covered and incubated for 30 min at room temperature on a plate shaker. Plates were then washed twice, and beads were resuspended in sheath fluid, placed on shaker for 5 min, and then read on a Bio‐Plex^® 200 following manufacturers’ specifications and analyzed using Bio‐Plex Manager software v6.0 (Bio‐Rad, Hercules, CA).